Journal articles: 'PLP-dependent enzyme'

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Ngo, Ho-Phuong-Thuy, Nuno M. F. S. A. Cerqueira, Jin-Kwang Kim, Myoung-Ki Hong, Pedro Alexandrino Fernandes, Maria João Ramos, and Lin-Woo Kang. "PLP undergoes conformational changes during the course of an enzymatic reaction." Acta Crystallographica Section D Biological Crystallography 70, no. 2 (January 31, 2014): 596–606. http://dx.doi.org/10.1107/s1399004713031283.

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Numerous enzymes, such as the pyridoxal 5′-phosphate (PLP)-dependent enzymes, require cofactors for their activities. Using X-ray crystallography, structural snapshots of the L-serine dehydratase catalytic reaction of a bacterial PLP-dependent enzyme were determined. In the structures, the dihedral angle between the pyridine ring and the Schiff-base linkage of PLP varied from 18° to 52°. It is proposed that the organic cofactor PLP directly catalyzes reactions by active conformational changes, and the novel catalytic mechanism involving the PLP cofactor was confirmed by high-level quantum-mechanical calculations. The conformational change was essential for nucleophilic attack of the substrate on PLP, for concerted proton transfer from the substrate to the protein and for directing carbanion formation of the substrate. Over the whole catalytic cycle, the organic cofactor catalyzes a series of reactions, like the enzyme. The conformational change of the PLP cofactor in catalysis serves as a starting point for identifying the previously unknown catalytic roles of organic cofactors.

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AL Mughram, Mohammed H., Mohini S. Ghatge, Glen E. Kellogg, and Martin K. Safo. "Elucidating the Interaction between Pyridoxine 5′-Phosphate Oxidase and Dopa Decarboxylase: Activation of B6-Dependent Enzyme." International Journal of Molecular Sciences 24, no. 1 (December 30, 2022): 642. http://dx.doi.org/10.3390/ijms24010642.

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Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, serves as a cofactor for scores of B6-dependent (PLP-dependent) enzymes involved in many cellular processes. One such B6 enzyme is dopa decarboxylase (DDC), which is required for the biosynthesis of key neurotransmitters, e.g., dopamine and serotonin. PLP-dependent enzymes are biosynthesized as apo-B6 enzymes and then converted to the catalytically active holo-B6 enzymes by Schiff base formation between the aldehyde of PLP and an active site lysine of the protein. In eukaryotes, PLP is made available to the B6 enzymes through the activity of the B6-salvage enzymes, pyridoxine 5′-phosphate oxidase (PNPO) and pyridoxal kinase (PLK). To minimize toxicity, the cell keeps the content of free PLP (unbound) very low through dephosphorylation and PLP feedback inhibition of PNPO and PLK. This has led to a proposed mechanism of complex formation between the B6-salvage enzymes and apo-B6 enzymes prior to the transfer of PLP, although such complexes are yet to be characterized at the atomic level, presumably due to their transient nature. A computational study, for the first time, was used to predict a likely PNPO and DDC complex, which suggested contact between the allosteric PLP tight-binding site on PNPO and the active site of DDC. Using isothermal calorimetry and/or surface plasmon resonance, we also show that PNPO binds both apoDDC and holoDDC with dissociation constants of 0.93 ± 0.07 μM and 2.59 ± 0.11 μM, respectively. Finally, in the presence of apoDDC, the tightly bound PLP on PNPO is transferred to apoDDC, resulting in the formation of about 35% holoDDC.

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Kawakami, Ryushi, Chinatsu Kinoshita, Tomoki Kawase, Mikio Sato, Junji Hayashi, Haruhiko Sakuraba, and Toshihisa Ohshima. "Characterization of a novel moderate-substrate specificity amino acid racemase from the hyperthermophilic archaeon Thermococcus litoralis." Bioscience, Biotechnology, and Biochemistry 85, no. 7 (May 4, 2021): 1650–57. http://dx.doi.org/10.1093/bbb/zbab078.

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ABSTRACT The amino acid sequence of the OCC_10945 gene product from the hyperthermophilic archaeon Thermococcus litoralis DSM5473, originally annotated as γ-aminobutyrate aminotransferase, is highly similar to that of the uncharacterized pyridoxal 5ʹ-phosphate (PLP)-dependent amino acid racemase from Pyrococcus horikoshii. The OCC_10945 enzyme was successfully overexpressed in Escherichia coli by coexpression with a chaperone protein. The purified enzyme demonstrated PLP-dependent amino acid racemase activity primarily toward Met and Leu. Although PLP contributed to enzyme stability, it only loosely bound to this enzyme. Enzyme activity was strongly inhibited by several metal ions, including Co2+ and Zn2+, and nonsubstrate amino acids such as l-Arg and l-Lys. These results suggest that the underlying PLP-binding and substrate recognition mechanisms in this enzyme are significantly different from those of the other archaeal and bacterial amino acid racemases. This is the first description of a novel PLP-dependent amino acid racemase with moderate substrate specificity in hyperthermophilic archaea.

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Zou, Lingling, Yang Song, Chengliang Wang, Jiaqi Sun, Leilei Wang, Beijiu Cheng, and Jun Fan. "Crystal structure of maize serine racemase with pyridoxal 5′-phosphate." Acta Crystallographica Section F Structural Biology Communications 72, no. 3 (February 16, 2016): 165–71. http://dx.doi.org/10.1107/s2053230x16000960.

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Serine racemase (SR) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that is responsible for D-serine biosynthesisin vivo. The first X-ray crystal structure of maize SR was determined to 2.1 Å resolution and PLP binding was confirmed in solution by UV–Vis absorption spectrometry. Maize SR belongs to the type II PLP-dependent enzymes and differs from the SR of a vancomycin-resistant bacterium. The PLP is bound to each monomer by forming a Schiff base with Lys67. Structural comparison with rat and fission yeast SRs reveals a similar arrangement of active-site residues but a different orientation of the C-terminal helix.

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Rocha, Juliana F., André F. Pina, Sérgio F. Sousa, and Nuno M. F. S. A. Cerqueira. "PLP-dependent enzymes as important biocatalysts for the pharmaceutical, chemical and food industries: a structural and mechanistic perspective." Catalysis Science & Technology 9, no. 18 (2019): 4864–76. http://dx.doi.org/10.1039/c9cy01210a.

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Yoshikane, Yu, Nana Yokochi, Kouhei Ohnishi, Hideyuki Hayashi, and Toshiharu Yagi. "Molecular cloning, expression and characterization of pyridoxamine–pyruvate aminotransferase." Biochemical Journal 396, no. 3 (May 29, 2006): 499–507. http://dx.doi.org/10.1042/bj20060078.

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Pyridoxamine–pyruvate aminotransferase is a PLP (pyridoxal 5′-phosphate) (a coenzyme form of vitamin B6)-independent aminotransferase which catalyses a reversible transamination reaction between pyridoxamine and pyruvate to form pyridoxal and L-alanine. The gene encoding the enzyme has been identified, cloned and overexpressed for the first time. The mlr6806 gene on the chromosome of a symbiotic nitrogen-fixing bacterium, Mesorhizobium loti, encoded the enzyme, which consists of 393 amino acid residues. The primary sequence was identical with those of archaeal aspartate aminotransferase and rat serine–pyruvate aminotransferase, which are PLP-dependent aminotransferases. The results of fold-type analysis and the consensus amino acid residues found around the active-site lysine residue identified in the present study showed that the enzyme could be classified into class V aminotransferases of fold type I or the AT IV subfamily of the α family of the PLP-dependent enzymes. Analyses of the absorption and CD spectra of the wild-type and point-mutated enzymes showed that Lys197 was essential for the enzyme activity, and was the active-site lysine residue that corresponded to that found in the PLP-dependent aminotransferases, as had been suggested previously [Hodsdon, Kolb, Snell and Cole (1978) Biochem. J. 169, 429–432]. The Kd value for pyridoxal determined by means of CD was 100-fold lower than the Km value for it, suggesting that Schiff base formation between pyridoxal and the active-site lysine residue is partially rate determining in the catalysis of pyridoxal. The active-site structure and evolutionary aspects of the enzyme are discussed.

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Williamson, P. R., J. M. Kittler, J. W. Thanassi, and H. M. Kagan. "Reactivity of a functional carbonyl moiety in bovine aortic lysyl oxidase. Evidence against pyridoxal 5′-phosphate." Biochemical Journal 235, no. 2 (April 15, 1986): 597–605. http://dx.doi.org/10.1042/bj2350597.

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Previous studies have pointed towards a cofactor role for pyridoxal 5′-phosphate (PLP) in lysyl oxidase, the enzyme that generates the peptidyl aldehyde precursor to the lysine-derived cross-linkages in elastin and collagen. The nature of a carbonyl moiety in purified bovine aortic lysyl oxidase was explored in the present study. A PLP dinitrophenylhydrazone could not be isolated from lysyl oxidase, although corresponding preparations of aspartate aminotransferase, a PLP-dependent enzyme, yielded this derivative, as revealed by h.p.l.c. Analysis of lysyl oxidase for PLP after reduction of the enzyme by NaBH4, a procedure that converts PLP-protein aldimines into stable 5′-phosphopyridoxyl functions, also proved negative in tests using monoclonal antibody specific for this epitope. Lysyl oxidase was competitively inhibited by phenylhydrazine, and inhibition became irreversible with time at 37 degrees C, displaying a first-order inactivation rate constant of 0.4 min-1 and KI of 1 microM. [14C]Phenylhydrazine was covalently incorporated into the enzyme in a manner that was prevented by prior modification of the enzyme with beta-aminopropionitrile, a specific active-site inhibitor, and which correlated with functional active-site content. The chemical stability of the enzyme-bound phenylhydrazine exceeded that expected of linkages between PLP and proteins. The absorption spectrum of the phenylhydrazine derivative of lysyl oxidase was clearly distinct from that of the phenylhydrazone of PLP. It is concluded that lysyl oxidase contains a carbonyl cofactor that is not identical with PLP and that is bound to the enzyme by a stable chemical bond.

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MOORE, Patrick S., Paola DOMINICI, and Carla BORRI VOLTATTORNI. "Cloning and expression of pig kidney dopa decarboxylase: comparison of the naturally occurring and recombinant enzymes." Biochemical Journal 315, no. 1 (April 1, 1996): 249–56. http://dx.doi.org/10.1042/bj3150249.

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L-Aromatic amino acid decarboxylase (dopa decarboxylase; DDC) is a pyridoxal 5´-phosphate (PLP)-dependent homodimeric enzyme that catalyses the decarboxylation of L-dopa and other L-aromatic amino acids. To advance structure–function studies with the enzyme, a cDNA that codes for the protein from pig kidney has been cloned by joining a partial cDNA obtained by library screening with a synthetic portion constructed by the annealing and extension of long oligonucleotides. The hybrid cDNA was then expressed in Escherichia coli to produce recombinant protein. During characterization of the recombinant enzyme it was unexpectedly observed that it possesses certain differences from the enzyme purified from pig kidney. Whereas the latter protein binds 1 molecule of PLP per dimer, the recombinant enzyme was found to bind two molecules of coenzyme per dimer. Moreover, the Vmax was twice that of the protein purified from tissue. On addition of substrate, the absorbance changes accompanying transaldimination were likewise 2-fold greater in the recombinant enzyme. Examination of the respective apoenzymes by absorbance, CD and fluorescence spectroscopy revealed distinct differences. The recombinant apoprotein has no significant absorbance at 335 nm, unlike the pig kidney apoenzyme; in the latter case this residual absorbance is associated with a positive dichroic signal. When excited at 335 nm the pig kidney apoenzyme has a pronounced emission maximum at 385 nm, in contrast with its recombinant counterpart, which shows a weak broad emission at about 400 nm. However, the holoenzyme–apoenzyme transition did not markedly alter the respective fluorescence properties of either recombinant or pig kidney DDC when excited at 335 nm. Taken together, these findings indicate that recombinant pig kidney DDC has two active-site PLP molecules and therefore displays structural characteristics typical of PLP-dependent homodimeric enzymes. The natural enzyme contains one active-site PLP molecule whereas the remaining PLP binding site is most probably occupied by an inactive covalently bound coenzyme derivative; some speculations are made about its origin. The coenzyme absorbing bands of recombinant DDC show a modest pH dependence at 335 and 425 nm. A putative working model is presented to explain this behaviour.

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Kezuka, Yuichiro, Yasuo Yoshida, and Takamasa Nonaka. "Structure of hydrogen sulfide-producing enzyme from a periodontal pathogen." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C454. http://dx.doi.org/10.1107/s205327331409545x.

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Hydrogen sulfide (H2S) is one of the predominant volatile sulfur compounds that are primarily responsible for oral malodor and contribute to the progress of periodontal disease. H2S in the human oral cavity is generally produced by enzymatic actions of oral bacteria.Fusobacterium nucleatum, a Gram negative periodontal pathogen, is known to be one of the heaviest H2S producers [1]. For now, four genes (fn0625,fn1055,fn1220, andfn1419) encoding pyridoxal-5′-phosphate (PLP)-dependent H2S-producing enzymes have been identified and characterized inF. nucleatumATCC 25586. Of the four enzymes, Fn1055 protein is a unique H2S-producing enzyme, which produces H2S and L-serine from L-cysteine [2]. Therefore, Fn1055 might play important roles in L-serine biosynthesis in addition to H2S production in this periodontal pathogen. Crystal structures of recombinant Fn1055 and its site-directed mutant complex with L-cysteine (a substrate) were determined at 2.1 Å resolution. The enzyme forms a homodimer whose subunits are related by a two-fold axis. The subunit is composed of two domains with α/β structure. The PLP cofactor forms a covalent internal aldimine linkage with the ε-amino group of Lys46 at the bottom of active site cleft between the domains, in the absence of substrate. On the other hand, in the cocrystal of mutant with L-cysteine, the introduced L-cysteine was found to be covalently bound to PLP, instead of Lys46. This covalent intermediate was identified as an α-aminoacrylate, which is the key species of PLP-dependent-enzyme catalysis, by spectrophotometric measurement. Along with the intermediate formation, closure of active site cleft was also observed. Furthermore, we found an amino acid residue acting as a base and confirmed its indispensability for catalysis by enzymatic analyses. These results support that H2S production by Fn1055 proceeds through the β-elimination of L-cysteine, and enable us to propose a detailed catalytic mechanism of Fn1055.

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Gao, Sisi, Huanting Liu, Valérie de Crécy-Lagard, Wen Zhu, Nigel G. J. Richards, and James H. Naismith. "PMP–diketopiperazine adducts form at the active site of a PLP dependent enzyme involved in formycin biosynthesis." Chemical Communications 55, no. 96 (2019): 14502–5. http://dx.doi.org/10.1039/c9cc06975e.

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Raasakka, Arne, Elaheh Mahootchi, Ingeborg Winge, Weisha Luan, Petri Kursula, and Jan Haavik. "Structure of the mouse acidic amino acid decarboxylase GADL1." Acta Crystallographica Section F Structural Biology Communications 74, no. 1 (January 1, 2018): 65–73. http://dx.doi.org/10.1107/s2053230x17017848.

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Pyridoxal 5′-phosphate (PLP) is a ubiquitous cofactor in various enzyme classes, including PLP-dependent decarboxylases. A recently discovered member of this class is glutamic acid decarboxylase-like protein 1 (GADL1), which lacks the activity to decarboxylate glutamate to γ-aminobutyrate, despite its homology to glutamic acid decarboxylase. Among the acidic amino acid decarboxylases, GADL1 is most similar to cysteine sulfinic acid decarboxylase (CSAD), but the physiological function of GADL1 is unclear, although its expression pattern and activity suggest a role in neurotransmitter and neuroprotectant metabolism. The crystal structure of mouse GADL1 is described, together with a solution model based on small-angle X-ray scattering data. While the overall fold and the conformation of the bound PLP are similar to those in other PLP-dependent decarboxylases, GADL1 adopts a more loose conformation in solution, which might have functional relevance in ligand binding and catalysis. The structural data raise new questions about the compactness, flexibility and conformational dynamics of PLP-dependent decarboxylases, including GADL1.

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Mizobuchi, Taichi, Risako Nonaka, Motoki Yoshimura, Katsumasa Abe, Shouji Takahashi, Yoshio Kera, and Masaru Goto. "Crystal structure of a pyridoxal 5′-phosphate-dependent aspartate racemase derived from the bivalve mollusc Scapharca broughtonii." Acta Crystallographica Section F Structural Biology Communications 73, no. 12 (November 6, 2017): 651–56. http://dx.doi.org/10.1107/s2053230x17015813.

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Aspartate racemase (AspR) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that is responsible for D-aspartate biosynthesis in vivo. To the best of our knowledge, this is the first study to report an X-ray crystal structure of a PLP-dependent AspR, which was resolved at 1.90 Å resolution. The AspR derived from the bivalve mollusc Scapharca broughtonii (SbAspR) is a type II PLP-dependent enzyme that is similar to serine racemase (SR) in that SbAspR catalyzes both racemization and dehydration. Structural comparison of SbAspR and SR shows a similar arrangement of the active-site residues and nucleotide-binding site, but a different orientation of the metal-binding site. Superposition of the structures of SbAspR and of rat SR bound to the inhibitor malonate reveals that Arg140 recognizes the β-carboxyl group of the substrate aspartate in SbAspR. It is hypothesized that the aromatic proline interaction between the domains, which favours the closed form of SbAspR, influences the arrangement of Arg140 at the active site.

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Deka, Geeta, Shveta Bisht, H. S. Savithri, and M. R. N. Murthy. "Structural studies on the catalytic mechanism of Diaminopropionate ammonia lyase." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1822. http://dx.doi.org/10.1107/s2053273314081789.

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Diaminopropionate ammonia lyase (DAPAL) is a non-stereo specific fold-type II pyridoxal 5' phosphate (PLP) dependent enzyme that catalyzes the conversion of both D/L isoforms of the nonstandard amino acid Diaminopropionate (DAP) to pyruvate and ammonia. DAP is important for the synthesis of nonribosomal peptide antibiotics such as viomycin and capreomycin. Earlier structural studies on EcDAPAL bound to a reaction intermediate (aminoacrylate) suggested that the enzyme follows a two base mechanism, where Asp120 and Lys77 function as general bases to abstract proton from D-DAP and L-DAP respectively. A novel disulfide was observed near the active site, although its functional significance was not clear. In the present study, structural and biochemical characterization of active site mutants Asp120 (Asp120Asn/Ser/Thr/Cys) and Lys77 (Lys77His/ Thr/Ala/Val) of EcDAPAL has been carried out. Reduction of catalytic efficiency (Kcat/Km) of D120N EcDAPAL for D-DAP by 140 fold and presence of the uncatalyzed ligand at the active site in the crystal structure suggested that Asp120 indeed abstracts proton from D-DAP. Lys77, which was speculated to be important for proton abstraction from L DAP, however seemed to be crucial for PLP binding only. Presence of non-covalently bound PLP in the L77W mutant and occurence of both the ketoenamine, enolimine forms of internal aldimine in L77R mutant provided an in depth insight into the unique chemistry of internal aldimine formation in PLP dependent enzymes. To investigate the role of the novel disulfide bond near the active site, C265 and C291 were mutated to Serine. Studies on these mutants show that this disulfide bond gives additional stability to the protein and might regulate the entry of substrates to the active site. Thus, these studies provide deeper insights into the reaction mechanism of EcDAPAL, representing the overall reaction mechanism followed by several other fold-type II PLP pendent enzymes.

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Borchert, Andrew J., Jacquelyn M. Walejko, Adrien Le Guennec, Dustin C. Ernst, Arthur S. Edison, and Diana M. Downs. "Integrated Metabolomics and Transcriptomics Suggest the Global Metabolic Response to 2-Aminoacrylate Stress in Salmonella enterica." Metabolites 10, no. 1 (December 24, 2019): 12. http://dx.doi.org/10.3390/metabo10010012.

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In Salmonella enterica, 2-aminoacrylate (2AA) is a reactive enamine intermediate generated during a number of biochemical reactions. When the 2-iminobutanoate/2-iminopropanoate deaminase (RidA; EC: 3.5.99.10) is eliminated, 2AA accumulates and inhibits the activity of multiple pyridoxal 5’-phosphate(PLP)-dependent enzymes. In this study, untargeted proton nuclear magnetic resonance (1H NMR) metabolomics and transcriptomics data were used to uncover the global metabolic response of S. enterica to the accumulation of 2AA. The data showed that elimination of RidA perturbed folate and branched chain amino acid metabolism. Many of the resulting perturbations were consistent with the known effect of 2AA stress, while other results suggested additional potential enzyme targets of 2AA-dependent damage. The majority of transcriptional and metabolic changes appeared to be the consequence of downstream effects on the metabolic network, since they were not directly attributable to a PLP-dependent enzyme. In total, the results highlighted the complexity of changes stemming from multiple perturbations of the metabolic network, and suggested hypotheses that will be valuable in future studies of the RidA paradigm of endogenous 2AA stress.

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Han, Qinghong, Mingxu Xu, Li Tang, Xuezhong Tan, Xiuying Tan, Yuying Tan, and Robert M. Hoffman. "Homogeneous, Nonradioactive, Enzymatic Assay for Plasma Pyridoxal 5-Phosphate." Clinical Chemistry 48, no. 9 (September 1, 2002): 1560–64. http://dx.doi.org/10.1093/clinchem/48.9.1560.

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Abstract Background: Pyridoxal 5′-phosphate (PLP) is the biologically active form of vitamin B6. Clinical studies suggest that low PLP concentrations are an independent risk factor for cardiovascular and other diseases. However, PLP concentrations are not routinely diagnosed because of the lack of a homogeneous, nonradioactive assay. We describe a homogeneous, nonradioactive, enzymatic PLP assay that uses the apo form of the PLP-dependent recombinant enzyme, homocysteine-α,γ-lyase (rHCYase). Methods: PLP was removed from holoenzyme rHCYase by incubation with hydroxylamine to obtain apo-rHCYase. The restoration of enzymatic activity by reconstitution of the holoenzyme was linearly related to the amount of PLP bound to the enzyme. The amplification principle of the assay allowed nanomolar concentrations of PLP to be measured by the conversion (by reconstituted holo-rHCYase) of millimolar concentrations of homocysteine to H2S. N,N-Dibutylphenylenediamine (DBPDA) was used for determination of H2S, the combination of which forms a chromophore with high absorbance. The assay was initiated by incubation of 5 μL of plasma with apo-rHCYase in a binding buffer for 60 min at 37 °C. Homocysteine (2.5 mmol/L) was added to the assay buffer and incubated at 37 °C for 20 min. The DBPDA reaction was allowed to progress for 10 min and then read at 675 nm. Results: The PLP enzymatic assay has a lower limit of detection of 5 nmol/L and is linear to 200 nmol/L. The recovery of PLP was 98%. The mean within- and between-run CVs were 9.6% and 12%, respectively. Correlation of 45 samples in the PLP enzymatic assay and the B63H radioenzymatic assay (American Laboratory Products Co., Ltd.) yielded: y = 0.9367x + 10.569 (R2 = 0.9201). Conclusions: This new PLP assay is the first homogeneous, nonradioactive, vitamin B6 diagnostic method. The assay is applicable to chemistry automated analyzers and may have wide clinical use.

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JALA, Venkatakrishna Rao, Naropantul APPAJI RAO, and Handanahal Subbarao SAVITHRI. "Identification of amino acid residues, essential for maintaining the tetrameric structure of sheep liver cytosolic serine hydroxymethyltransferase, by targeted mutagenesis." Biochemical Journal 369, no. 3 (February 1, 2003): 469–76. http://dx.doi.org/10.1042/bj20021160.

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Serine hydroxymethyltransferase (SHMT), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, catalyses the transfer of the hydroxymethyl group from serine to tetrahydrofolate to yield glycine and N5,N10-methylenetetrahydrofolate. An analysis of the known SHMT sequences indicated that several amino acid residues were conserved. In this paper, we report the identification of the amino acid residues essential for maintaining the oligomeric structure of sheep liver cytosolic recombinant SHMT (scSHMT) through intra- and inter-subunit interactions and by stabilizing the binding of PLP at the active site. The mutation of Lys-71, Arg-80 and Asp-89, the residues involved in intra-subunit ionic interactions, disturbed the oligomeric structure and caused a loss of catalytic activity. Mutation of Trp-110 to Phe was without effect, while its mutation to Ala resulted in the enzyme being present in the insoluble fraction. These results suggested that Trp-110 located in a cluster of hydrophobic residues was essential for proper folding of the enzyme. Arg-98 and His-304, residues involved in the inter-subunit interactions, were essential for maintaining the tetrameric structure. Mutation of Tyr-72, Asp-227 and His-356 at the active site which interact with PLP resulted in the loss of PLP, and hence loss of tetrameric structure. Mutation of Cys-203, located away from the active site, weakened PLP binding indirectly. The results demonstrate that in addition to residues involved in inter-subunit interactions, those involved in PLP binding and intra-subunit interactions also affect the oligomeric structure of scSHMT.

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Campanini, Barbara, Stefano Bettati, Martino Luigi di Salvo, Andrea Mozzarelli, and Roberto Contestabile. "Asymmetry of the Active Site Loop Conformation between Subunits of Glutamate-1-semialdehyde Aminomutase in Solution." BioMed Research International 2013 (2013): 1–10. http://dx.doi.org/10.1155/2013/353270.

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Glutamate-1-semialdehyde aminomutase (GSAM) is a dimeric, pyridoxal 5′-phosphate (PLP)- dependent enzyme catalysing in plants and some bacteria the isomerization of L-glutamate-1-semialdehyde to 5-aminolevulinate, a common precursor of chlorophyll, haem, coenzyme B12, and other tetrapyrrolic compounds. During the catalytic cycle, the coenzyme undergoes conversion from pyridoxamine 5′-phosphate (PMP) to PLP. The entrance of the catalytic site is protected by a loop that is believed to switch from an open to a closed conformation during catalysis. Crystallographic studies indicated that the structure of the mobile loop is related to the form of the cofactor bound to the active site, allowing for asymmetry within the dimer. Since no information on structural and functional asymmetry of the enzyme in solution is available in the literature, we investigated the active site accessibility by determining the cofactor fluorescence quenching of PMP- and PLP-GSAM forms. PLP-GSAM is partially quenched by potassium iodide, suggesting that at least one catalytic site is accessible to the anionic quencher and therefore confirming the asymmetry observed in the crystal structure. Iodide induces release of the cofactor from PMP-GSAM, apparently from only one catalytic site, therefore suggesting an asymmetry also in this form of the enzyme in solution, in contrast with the crystallographic data.

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Deshmukh, Ashish, and Balasubramanian Gopal. "Structural insights into the catalytic mechanism of Bacillus subtilis BacF." Acta Crystallographica Section F Structural Biology Communications 76, no. 3 (March 1, 2020): 145–51. http://dx.doi.org/10.1107/s2053230x20001636.

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The nonribosomal biosynthesis of the dipeptide antibiotic bacilysin is achieved by the concerted action of multiple enzymes in the Bacillus subtilis bac operon. BacF (YwfG), encoded by the bacF gene, is a fold type I pyridoxal 5-phosphate (PLP)-dependent stereospecific transaminase. Activity assays with L-phenylalanine and 4-hydroxyphenylpyruvic acid (4HPP), a chemical analogue of tetrahydrohydroxyphenylpyruvic acid (H4HPP), revealed stereospecific substrate preferences, a finding that was consistent with previous reports on the role of this enzyme in bacilysin synthesis. The crystal structure of this dimeric enzyme was determined in its apo form as well as in substrate-bound and product-bound conformations. Two ligand-bound structures were determined by soaking BacF crystals with substrates (L-phenylalanine and 4-hydroxyphenylpyruvate). These structures reveal multiple catalytic steps: the internal aldimine with PLP and two external aldimine conformations that show the rearrangement of the external aldimine to generate product (L-tyrosine). Together, these structural snapshots provide an insight into the catalytic mechanism of this transaminase.

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Cellini, Barbara, Mariarita Bertoldi, Riccardo Montioli, Alessandro Paiardini, and Carla Borri Voltattorni. "Human wild-type alanine:glyoxylate aminotransferase and its naturally occurring G82E variant: functional properties and physiological implications." Biochemical Journal 408, no. 1 (October 29, 2007): 39–50. http://dx.doi.org/10.1042/bj20070637.

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Human hepatic peroxisomal AGT (alanine:glyoxylate aminotransferase) is a PLP (pyridoxal 5′-phosphate)-dependent enzyme whose deficiency causes primary hyperoxaluria Type I, a rare autosomal recessive disorder. To acquire experimental evidence for the physiological function of AGT, the Keq,overall of the reaction, the steady-state kinetic parameters of the forward and reverse reactions, and the pre-steady-state kinetics of the half-reactions of the PLP form of AGT with L-alanine or glycine and the PMP (pyridoxamine 5′-phosphate) form with pyruvate or glyoxylate have been measured. The results indicate that the enzyme is highly specific for catalysing glyoxylate to glycine processing, thereby playing a key role in glyoxylate detoxification. Analysis of the reaction course also reveals that PMP remains bound to the enzyme during the catalytic cycle and that the AGT–PMP complex displays a reactivity towards oxo acids higher than that of apoAGT in the presence of PMP. These findings are tentatively related to possible subtle rearrangements at the active site also indicated by the putative binding mode of catalytic intermediates. Additionally, the catalytic and spectroscopic features of the naturally occurring G82E variant have been analysed. Although, like the wild-type, the G82E variant is able to bind 2 mol PLP/dimer, it exhibits a significant reduced affinity for PLP and even more for PMP compared with wild-type, and an altered conformational state of the bound PLP. The striking molecular defect of the mutant, consisting in the dramatic decrease of the overall catalytic activity (∼0.1% of that of normal AGT), appears to be related to the inability to undergo an efficient transaldimination of the PLP form of the enzyme with amino acids as well as an efficient conversion of AGT–PMP into AGT–PLP. Overall, careful biochemical analyses have allowed elucidation of the mechanism of action of AGT and the way in which the disease causing G82E mutation affects it.

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Steffen-Munsberg, Fabian, Clare Vickers, Hannes Kohls, Henrik Land, Hendrik Mallin, Alberto Nobili, Lilly Skalden, et al. "Bioinformatic analysis of a PLP-dependent enzyme superfamily suitable for biocatalytic applications." Biotechnology Advances 33, no. 5 (September 2015): 566–604. http://dx.doi.org/10.1016/j.biotechadv.2014.12.012.

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Mukherjee, Mandira, Stuart A. Sievers, Mark T. Brown, and Patricia J. Johnson. "Identification and Biochemical Characterization of Serine Hydroxymethyl Transferase in the Hydrogenosome of Trichomonas vaginalis." Eukaryotic Cell 5, no. 12 (September 15, 2006): 2072–78. http://dx.doi.org/10.1128/ec.00249-06.

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ABSTRACT Serine hydroxymethyl transferase (SHMT) is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. We have identified a single gene encoding SHMT in the genome of Trichomonas vaginalis, an amitochondriate, deep-branching unicellular protist. The protein possesses a putative N-terminal hydrogenosomal presequence and was shown to localize to hydrogensomes by immunofluorescence analysis, providing evidence of amino acid metabolism in this unusual organelle. In contrast to the tetrameric SHMT that exists in the mammalian host, we found that the T. vaginalis SHMT is a homodimer, as found in prokaryotes. All examined SHMT contain an 8-amino-acid conserved sequence, VTTTTHKT, containing the active-site lysyl residue (Lys 251 in TvSHMT) that forms an internal aldimine with PLP. We mutated this Lys residue to Arg and Gln and examined structural and catalytic properties of the wild-type and mutant enzymes in comparison to that reported for the mammalian protein. The oligomeric structure of the mutant K251R and K251Q TvSHMT was not affected, in contrast to that observed for comparable mutations in the mammalian enzyme. Likewise, contrary to that observed for mammalian SHMT, the catalytic activity of K251R TvSHMT was unaffected in the presence of PLP. The K251Q TvSHMT, however, was found to be inactive. These studies indicate that the active site of the parasite enzyme is distinct from its prokaryotic and eukaryotic counterparts and identify TvSHMT as a potential drug target.

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Ikushiro, Hiroko, Mohammad Mainul Islam, Hiromasa Tojo, and Hideyuki Hayashi. "Molecular Characterization of Membrane-Associated Soluble Serine Palmitoyltransferases from Sphingobacterium multivorum and Bdellovibrio stolpii." Journal of Bacteriology 189, no. 15 (June 8, 2007): 5749–61. http://dx.doi.org/10.1128/jb.00194-07.

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ABSTRACT Serine palmitoyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of l-serine and palmitoyl coenzyme A (CoA) to form 3-ketodihydrosphingosine (KDS). Eukaryotic SPTs comprise tightly membrane-associated heterodimers belonging to the pyridoxal 5′-phosphate (PLP)-dependent α-oxamine synthase family. Sphingomonas paucimobilis, a sphingolipid-containing bacterium, contains an abundant water-soluble homodimeric SPT of the same family (H. Ikushiro et al., J. Biol. Chem. 276:18249-18256, 2001). This enzyme is suitable for the detailed mechanistic studies of SPT, although single crystals appropriate for high-resolution crystallography have not yet been obtained. We have now isolated three novel SPT genes from Sphingobacterium multivorum, Sphingobacterium spiritivorum, and Bdellovibrio stolpii, respectively. Each gene product exhibits an ∼30% sequence identity to both eukaryotic subunits, and the putative catalytic amino acid residues are conserved. All bacterial SPTs were successfully overproduced in Escherichia coli and purified as water-soluble active homodimers. The spectroscopic properties of the purified SPTs are characteristic of PLP-dependent enzymes. The KDS formation by the bacterial SPTs was confirmed by high-performance liquid chromatography/mass spectrometry. The Sphingobacterium SPTs obeyed normal steady-state ordered Bi-Bi kinetics, while the Bdellovibrio SPT underwent a remarkable substrate inhibition at palmitoyl CoA concentrations higher than 100 μM, as does the eukaryotic enzyme. Immunoelectron microscopy showed that unlike the cytosolic Sphingomonas SPT, S. multivorum and Bdellovibrio SPTs were bound to the inner membrane of cells as peripheral membrane proteins, indicating that these enzymes can be a prokaryotic model mimicking the membrane-associated eukaryotic SPT.

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Mirzaei, Mitra, and Per Berglund. "Engineering of ωTransaminase for Effective Production of Chiral Amines." Journal of Computational and Theoretical Nanoscience 17, no. 6 (June 1, 2020): 2827–32. http://dx.doi.org/10.1166/jctn.2020.8947.

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ωTransaminases are pyridoxal-5-phosphat (PLP) dependent enzymes having the ability to catalyze the transference of an amino group to a keto compound. These enzymes are used for production of chiral amines which are important building blocks in pharmaceutical industry. There is often a need to improve enzyme properties such as enzyme stability, enzyme specificity and to decrease substrate-product inhibition. Here, protein engineering was applied to improve the enzyme activity of the enzyme from Chromobacterium violaceum Rational-design and site-directed mutagenesis were applied on position of (W60) in the active site of the enzyme. Different mutated enzyme variants such as W60H, W60F and W60Y were made. Also, the enantiopreference of the wild type enzyme was reversed to produce (R)-chiral amines. For this aim, a screening assay was followed by semi-rational approach and saturation mutagenesis in the active site of the enzyme. Creating the mutated enzyme libraries resulted to obtaining two enzyme variants. Their properties were low enantiopreference towards formations of (R)-enantiopreference and low specific constant ratio between fast and slow enantiomers (Evalue around one).

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Kasaragod, Vikram Babu, Anabel Pacios-Michelena, Natascha Schaefer, Fang Zheng, Nicole Bader, Christian Alzheimer, Carmen Villmann, and Hermann Schindelin. "Pyridoxal kinase inhibition by artemisinins down-regulates inhibitory neurotransmission." Proceedings of the National Academy of Sciences 117, no. 52 (December 14, 2020): 33235–45. http://dx.doi.org/10.1073/pnas.2008695117.

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The antimalarial artemisinins have also been implicated in the regulation of various cellular pathways including immunomodulation of cancers and regulation of pancreatic cell signaling in mammals. Despite their widespread application, the cellular specificities and molecular mechanisms of target recognition by artemisinins remain poorly characterized. We recently demonstrated how these drugs modulate inhibitory postsynaptic signaling by direct binding to the postsynaptic scaffolding protein gephyrin. Here, we report the crystal structure of the central metabolic enzyme pyridoxal kinase (PDXK), which catalyzes the production of the active form of vitamin B6 (also known as pyridoxal 5′-phosphate [PLP]), in complex with artesunate at 2.4-Å resolution. Partially overlapping binding of artemisinins with the substrate pyridoxal inhibits PLP biosynthesis as demonstrated by kinetic measurements. Electrophysiological recordings from hippocampal slices and activity measurements of glutamic acid decarboxylase (GAD), a PLP-dependent enzyme synthesizing the neurotransmitter γ-aminobutyric acid (GABA), define how artemisinins also interfere presynaptically with GABAergic signaling. Our data provide a comprehensive picture of artemisinin-induced effects on inhibitory signaling in the brain.

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Dajnowicz, Steven, Matthew Blakeley, David Keen, Andrey Kovalevsky, and Timothy Mueser. "Direct observation of protonation states in a PLP-dependent enzyme by neutron crystallography." Acta Crystallographica Section A Foundations and Advances 73, a1 (May 26, 2017): a26. http://dx.doi.org/10.1107/s0108767317099731.

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MUHAMMAD, MURTALA, YANGYANG LI, SIYU GONG, YANMIN SHI, JIANSONG JU, BAOHUA ZHAO, and DONG LIU. "Purification, Characterization and Inhibition of Alanine Racemase from a Pathogenic Strain of Streptococcus iniae." Polish Journal of Microbiology 68, no. 3 (September 2019): 331–41. http://dx.doi.org/10.33073/pjm-2019-036.

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Streptococcus iniae is a pathogenic and zoonotic bacteria that impacted high mortality to many fish species as well as capable of causing serious disease to humans. Alanine racemase (Alr, EC 5.1.1.1) is a pyridoxal-5’-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-alanine and D-alanine. In this study, we purified alanine racemase from S. iniae that was isolated from an infected Chinese sturgeon (Acipenser sinensis), as well as determined its biochemical characteristics and inhibitors. The alr gene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The enzyme has optimal activity at a temperature of 35°C and a pH of 9.5. It belongs to the PLP-dependent enzymes family and is highly specific to L-alanine. S.iniaeAlr (SiAlr) could be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters Km and Vmax of the enzyme were 33.11 mM, 2426 units/mg for L-alanine, and 14.36 mM, 963.6 units/mg for D-alanine. Finally, the 50% inhibitory concentrations (IC50) values and antibiotic activity of two alanine racemase inhibitors (homogentisic acid and hydroquinone), were determined and found to be effective against both Gram-positive and Gram-negative bacteria employed in this study.

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Beattie, Ashley E., Sita D. Gupta, Lenka Frankova, Agne Kazlauskaite, Jeffrey M. Harmon, Teresa M. Dunn, and Dominic J. Campopiano. "The Pyridoxal 5′-Phosphate (PLP)-Dependent Enzyme Serine Palmitoyltransferase (SPT): Effects of the Small Subunits and Insights from Bacterial Mimics of Human hLCB2a HSAN1 Mutations." BioMed Research International 2013 (2013): 1–13. http://dx.doi.org/10.1155/2013/194371.

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The pyridoxal 5′-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) catalyses the first step ofde novosphingolipid biosynthesis. The core human enzyme is a membrane-bound heterodimer composed of two subunits (hLCB1 and hLCB2a/b), and mutations in both hLCB1 (e.g., C133W and C133Y) and hLCB2a (e.g., V359M, G382V, and I504F) have been identified in patients with hereditary sensory and autonomic neuropathy type I (HSAN1), an inherited disorder that affects sensory and autonomic neurons. These mutations result in substrate promiscuity, leading to formation of neurotoxic deoxysphingolipids found in affected individuals. Here we measure the activities of the hLCB2a mutants in the presence of ssSPTa and ssSPTb and find that all decrease enzyme activity. High resolution structural data of the homodimeric SPT enzyme from the bacteriumSphingomonas paucimobilis(SpSPT) provides a model to understand the impact of the hLCB2a mutations on the mechanism of SPT. The three human hLCB2a HSAN1 mutations map ontoSpSPT (V246M, G268V, and G385F), and these mutant mimics reveal that the amino acid changes have varying impacts; they perturb the PLP cofactor binding, reduce the affinity for both substrates, decrease the enzyme activity, and, in the most severe case, cause the protein to be expressed in an insoluble form.

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Asojo, Oluwatoyin A., Sarah K. Nelson, Sara Mootien, Yashang Lee, Wanderson C. Rezende, Daniel A. Hyman, Monica M. Matsumoto, et al. "Structural and biochemical analyses of alanine racemase from the multidrug-resistantClostridium difficilestrain 630." Acta Crystallographica Section D Biological Crystallography 70, no. 7 (June 29, 2014): 1922–33. http://dx.doi.org/10.1107/s1399004714009419.

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Clostridium difficile, a Gram-positive, spore-forming anaerobic bacterium, is the leading cause of infectious diarrhea among hospitalized patients.C. difficileis frequently associated with antibiotic treatment, and causes diseases ranging from antibiotic-associated diarrhea to life-threatening pseudomembranous colitis. The severity ofC. difficileinfections is exacerbated by the emergence of hypervirulent and multidrug-resistant strains, which are difficult to treat and are often associated with increased mortality rates. Alanine racemase (Alr) is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that catalyzes the reversible racemization of L- and D-alanine. Since D-alanine is an essential component of the bacterial cell-wall peptidoglycan, and there are no known Alr homologs in humans, this enzyme is being tested as an antibiotic target. Cycloserine is an antibiotic that inhibits Alr. In this study, the catalytic properties and crystal structures of recombinant Alr from the virulent and multidrug-resistantC. difficilestrain 630 are presented. Three crystal structures ofC. difficileAlr (CdAlr), corresponding to the complex with PLP, the complex with cycloserine and a K271T mutant form of the enzyme with bound PLP, are presented. The structures are prototypical Alr homodimers with two active sites in which the cofactor PLP and cycloserine are localized. Kinetic analyses reveal that the K271T mutant CdAlr has the highest catalytic constants reported to date for any Alr. Additional studies are needed to identify the basis for the high catalytic activity. The structural and activity data presented are first steps towards using CdAlr for the development of structure-based therapeutics forC. difficileinfections.

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Dai, Guang Zhi, Wen Bo Han, Ya Ning Mei, Kuang Xu, Rui Hua Jiao, Hui Ming Ge, and Ren Xiang Tan. "Pyridoxal-5′-phosphate–dependent bifunctional enzyme catalyzed biosynthesis of indolizidine alkaloids in fungi." Proceedings of the National Academy of Sciences 117, no. 2 (December 27, 2019): 1174–80. http://dx.doi.org/10.1073/pnas.1914777117.

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Indolizidine alkaloids such as anticancer drugs vinblastine and vincristine are exceptionally attractive due to their widespread occurrence, prominent bioactivity, complex structure, and sophisticated involvement in the chemical defense for the producing organisms. However, the versatility of the indolizidine alkaloid biosynthesis remains incompletely addressed since the knowledge about such biosynthetic machineries is only limited to several representatives. Herein, we describe the biosynthetic gene cluster (BGC) for the biosynthesis of curvulamine, a skeletally unprecedented antibacterial indolizidine alkaloid from Curvularia sp. IFB-Z10. The molecular architecture of curvulamine results from the functional collaboration of a highly reducing polyketide synthase (CuaA), a pyridoxal-5′-phosphate (PLP)-dependent aminotransferase (CuaB), an NADPH-dependent dehydrogenase (CuaC), and a FAD-dependent monooxygenase (CuaD), with its transportation and abundance regulated by a major facilitator superfamily permease (CuaE) and a Zn(II)Cys6 transcription factor (CuaF), respectively. In contrast to expectations, CuaB is bifunctional and capable of catalyzing the Claisen condensation to form a new C–C bond and the α-hydroxylation of the alanine moiety in exposure to dioxygen. Inspired and guided by the distinct function of CuaB, our genome mining effort discovers bipolamines A−I (bipolamine G is more antibacterial than curvulamine), which represent a collection of previously undescribed polyketide alkaloids from a silent BGC in Bipolaris maydis ATCC48331. The work provides insight into nature’s arsenal for the indolizidine-coined skeletal formation and adds evidence in support of the functional versatility of PLP-dependent enzymes in fungi.

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Chen, Hao-Ping, Chin-Fen Lin, Ya-Jung Lee, San-San Tsay, and Shih-Hsiung Wu. "Purification and Properties of Ornithine Racemase from Clostridium sticklandii." Journal of Bacteriology 182, no. 7 (April 1, 2000): 2052–54. http://dx.doi.org/10.1128/jb.182.7.2052-2054.2000.

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ABSTRACT Ornithine racemase has been purified to homogeneity fromClostridium sticklandii, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This is the first racemase known to be highly specific to ornithine. This PLP-dependent enzyme has an M r of 92,000, with aKm for l-ornithine of 0.77 ± 0.05 mM and a kcat of 980 ± 20 s−1.

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Gaskin, Peter J., Harriet J. Adcock, Lorraine D. Buckberry, Paul H. Teesdale-Spittle, and P. Nicholas Shawl. "The C-S lysis of L-cysteine conjugates by aspartate and alanine aminotransferase enzymes." Human & Experimental Toxicology 14, no. 5 (May 1995): 422–27. http://dx.doi.org/10.1177/096032719501400506.

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One biotransformation pathway which is responsible for the generation of mutagenic and cytotoxic metabolites is that of the C-S lysis (CSL) of L-cysteine conjugates. Thirteen cysteine S-conjugates, synthesised in our labora tories, were incubated with porcine heart aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT), and the C-S lyase activity for each enzyme-sub strate combination was determined. ASAT and ALAT were shown to exhibit CSL activity. It was also demonstrated that this activity was inhibited in the presence of the pyri doxal phosphate (PLP)-dependent enzyme inhibitor amino(oxyacetic acid) (AOAA) confirming the pyridoxal phosphate dependent mechanism by which C-S lysis is known to take place. Since the activities of these enzymes are used as biomarkers for the assessment of organ dam age, the potential interaction of L-cysteine conjugates with them may suppress their activity through direct inhibition.

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Vozdek, Roman, Aleš Hnízda, Jakub Krijt, Marta Kostrouchová, and Viktor Kožich. "Novel structural arrangement of nematode cystathionine β-synthases: characterization of Caenorhabditis elegans CBS-1." Biochemical Journal 443, no. 2 (March 27, 2012): 535–47. http://dx.doi.org/10.1042/bj20111478.

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CBSs (cystathionine β-synthases) are eukaryotic PLP (pyridoxal 5 *-phosphate)-dependent proteins that maintain cellular homocysteine homoeostasis and produce cystathionine and hydrogen sulfide. In the present study, we describe a novel structural arrangement of the CBS enzyme encoded by the cbs-1 gene of the nematode Caenorhabditis elegans. The CBS-1 protein contains a unique tandem repeat of two evolutionarily conserved catalytic regions in a single polypeptide chain. These repeats include a catalytically active C-terminal module containing a PLP-binding site and a less conserved N-terminal module that is unable to bind the PLP cofactor and cannot catalyse CBS reactions, as demonstrated by analysis of truncated variants and active-site mutant proteins. In contrast with other metazoan enzymes, CBS-1 lacks the haem and regulatory Bateman domain essential for activation by AdoMet (S-adenosylmethionine) and only forms monomers. We determined the tissue and subcellular distribution of CBS-1 and showed that cbs-1 knockdown by RNA interference leads to delayed development and to an approximately 10-fold elevation of homocysteine concentrations in nematode extracts. The present study provides the first insight into the metabolism of sulfur amino acids and hydrogen sulfide in C. elegans and shows that nematode CBSs possess a structural feature that is unique among CBS proteins.

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BERTOLDI, Mariarita, Barbara CELLINI, Alessandro PAIARDINI, Martino Di SALVO, and Carla BORRIVOLTATTORNI. "Treponema denticola cystalysin exhibits significant alanine racemase activity accompanied by transamination: mechanistic implications1." Biochemical Journal 371, no. 2 (April 15, 2003): 473–83. http://dx.doi.org/10.1042/bj20020875.

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To obtain information on the reaction specificity of cystalysin from the spirochaete bacterium Treponema denticola, the interaction with l- and d-alanine has been investigated. Binding of both alanine enantiomers leads to the appearance of an external aldimine absorbing at 429nm and of a band absorbing at 498nm, indicative of a quinonoid species. Racemization and transamination reactions were observed to occur with both alanine isomers as substrates. The steady-state kinetic parameters for racemization, kcat and Km, for l-alanine are 1.05±0.03s−1 and 10±1mM respectively, whereas those for d-alanine are 1.4±0.1s−1 and 10±1mM. During the reaction of cystalysin with l- or d-alanine, a time-dependent loss of β-elimination activity occurs concomitantly with the conversion of the pyridoxal 5′-phosphate (PLP) coenzyme into pyridoxamine 5′-phosphate (PMP). The catalytic efficiency of the half-transamination of l-alanine is found to be 5.3×10−5 mM−1·s−1, 5-fold higher when compared with that of d-alanine. The partition ratio between racemization and half-transamination reactions is 2.3×103 for l-alanine and 1.4×104 for d-alanine. The pH dependence of the kinetic parameters for both the reactions shows that the enzyme possesses a single ionizing residue with pK values of 6.5–6.6, which must be unprotonated for catalysis. Addition of pyruvate converts the PMP form of the enzyme back into the PLP form and causes the concomitant recovery of β-elimination activity. In contrast with other PLP enzymes studied so far, but similar to alanine racemases, the apoform of the enzyme abstracted tritium from C4′ of both (4′S)- and (4′R)-[4′-3H]PMP in the presence of pyruvate. Together with molecular modelling of the putative binding sites of l- and d-alanine at the active site of the enzyme, the implications of these studies for the mechanisms of the side reactions catalysed by cystalysin are discussed.

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Sato, Dan, Tomoo Shiba, Sae Mizuno, Ayaka Kawamura, Shoko Hanada, Tetsuya Yamada, Mai Shinozaki, et al. "The hyperthermophilic cystathionine γ-synthase from the aerobic crenarchaeonSulfolobus tokodaii: expression, purification, crystallization and structural insights." Acta Crystallographica Section F Structural Biology Communications 73, no. 3 (February 21, 2017): 152–58. http://dx.doi.org/10.1107/s2053230x17002011.

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Cystathionine γ-synthase (CGS; EC 2.5.1.48), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, catalyzes the formation of cystathionine from an L-homoserine derivative and L-cysteine in the first step of the transsulfuration pathway. Recombinant CGS from the thermoacidophilic archaeonSulfolobus tokodaii(StCGS) was overexpressed inEscherichia coliand purified to homogeneity by heat treatment followed by hydroxyapatite and gel-filtration column chromatography. The purified enzyme shows higher enzymatic activity at 353 K under basic pH conditions compared with that at 293 K. Crystallization trials yielded three crystal forms from different temperature and pH conditions. Form I crystals (space groupP21; unit-cell parametersa= 58.4,b= 149.3,c= 90.2 Å, β = 108.9°) were obtained at 293 K under acidic pH conditions using 2-methyl-2,4-pentanediol as a precipitant, whereas under basic pH conditions the enzyme crystallized in form II at 293 K (space groupC2221; unit-cell parametersa= 117.7,b= 117.8,c= 251.3 Å) and in form II′ at 313 K (space groupC2221; unit-cell parametersa= 107.5,b= 127.7,c= 251.1 Å) using polyethylene glycol 3350 as a precipitant. X-ray diffraction data were collected to 2.2, 2.9 and 2.7 Å resolution for forms I, II and II′, respectively. Structural analysis of these crystal forms shows that the orientation of the bound PLP in form II is significantly different from that in form II′, suggesting that the change in orientation of PLP with temperature plays a role in the thermophilic enzymatic activity of StCGS.

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Yu, Xin-Jun, Chang-Yi Huang, Xiao-Dan Xu, Hong Chen, Miao-Jie Liang, Zhe-Xian Xu, Hui-Xia Xu, and Zhao Wang. "Protein Engineering of a Pyridoxal-5′-Phosphate-Dependent l-Aspartate-α-Decarboxylase from Tribolium castaneum for β-Alanine Production." Molecules 25, no. 6 (March 12, 2020): 1280. http://dx.doi.org/10.3390/molecules25061280.

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In the present study, a pyridoxal-5′-phosphate (PLP)-dependent L-aspartate-α-decarboxylase from Tribolium castaneum (TcPanD) was selected for protein engineering to efficiently produce β-alanine. A mutant TcPanD-R98H/K305S with a 2.45-fold higher activity than the wide type was selected through error-prone PCR, site-saturation mutagenesis, and 96-well plate screening technologies. The characterization of purified enzyme TcPanD-R98H/K305S showed that the optimal cofactor PLP concentration, temperature, and pH were 0.04% (m/v), 50 °C, and 7.0, respectively. The 1mM of Na+, Ni2+, Co2+, K+, and Ca2+ stimulated the activity of TcPanD-R98H/K305S, while only 5 mM of Ni2+ and Na+ could increase its activity. The kinetic analysis indicated that TcPanD-R98H/K305S had a higher substrate affinity and enzymatic reaction rate than the wild enzyme. A total of 267 g/L substrate l-aspartic acid was consumed and 170.5 g/L of β-alanine with a molar conversion of 95.5% was obtained under the optimal condition and 5-L reactor fermentation.

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Bakunova, Alina K., Alena Yu Nikolaeva, Tatiana V. Rakitina, Tatiana Y. Isaikina, Maria G. Khrenova, Konstantin M. Boyko, Vladimir O. Popov, and Ekaterina Yu Bezsudnova. "The Uncommon Active Site of D-Amino Acid Transaminase from Haliscomenobacter hydrossis: Biochemical and Structural Insights into the New Enzyme." Molecules 26, no. 16 (August 20, 2021): 5053. http://dx.doi.org/10.3390/molecules26165053.

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Among industrially important pyridoxal-5’-phosphate (PLP)-dependent transaminases of fold type IV D-amino acid transaminases are the least studied. However, the development of cascade enzymatic processes, including the synthesis of D-amino acids, renewed interest in their study. Here, we describe the identification, biochemical and structural characterization of a new D-amino acid transaminase from Haliscomenobacter hydrossis (Halhy). The new enzyme is strictly specific towards D-amino acids and their keto analogs; it demonstrates one of the highest rates of transamination between D-glutamate and pyruvate. We obtained the crystal structure of the Halhy in the holo form with the protonated Schiff base formed by the K143 and the PLP. Structural analysis revealed a novel set of the active site residues that differ from the key residues forming the active sites of the previously studied D-amino acids transaminases. The active site of Halhy includes three arginine residues, one of which is unique among studied transaminases. We identified critical residues for the Halhy catalytic activity and suggested functions of the arginine residues based on the comparative structural analysis, mutagenesis, and molecular modeling simulations. We suggested a strong positive charge in the O-pocket and the unshaped P-pocket as a structural code for the D-amino acid specificity among transaminases of PLP fold type IV. Characteristics of Halhy complement our knowledge of the structural basis of substrate specificity of D-amino acid transaminases and the sequence-structure-function relationships in these enzymes.

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Sköldberg, Filip, Fredrik Rorsman, Jaakko Perheentupa, Mona Landin-Olsson, Eystein S. Husebye, Jan Gustafsson, and Olle Kämpe. "Analysis of Antibody Reactivity against Cysteine Sulfinic Acid Decarboxylase, A Pyridoxal Phosphate-Dependent Enzyme, in Endocrine Autoimmune Disease." Journal of Clinical Endocrinology & Metabolism 89, no. 4 (April 1, 2004): 1636–40. http://dx.doi.org/10.1210/jc.2003-031161.

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Abstract The structurally related group II pyridoxal phosphate (PLP)-dependent amino acid decarboxylases glutamic acid decarboxylase (GAD), aromatic l-amino acid decarboxylase (AADC), and histidine decarboxylase (HDC) are known autoantigens in endocrine disorders. We report, for the first time, the prevalence of serum autoantibody reactivity against cysteine sulfinic acid decarboxylase (CSAD), an enzyme that shares 50% amino acid identity with the 65- and 67-kDa isoforms of GAD (GAD-65 and GAD-67), in endocrine autoimmune disease. Three of 83 patients (3.6%) with autoimmune polyendocrine syndrome type 1 (APS1) were anti-CSAD positive in a radioimmunoprecipitation assay. Anti-CSAD antibodies cross-reacted with GAD-65, and the anti-CSAD-positive sera were also reactive with AADC and HDC. The low frequency of anti-CSAD reactivity is in striking contrast to the prevalence of antibodies against GAD-65, AADC, and HDC in APS1 patients, suggesting that different mechanisms control the immunological tolerance toward CSAD and the other group II decarboxylases. Moreover, CSAD may be a useful mold for the construction of recombinant chimerical antigens in attempts to map conformational epitopes on other group II PLP-dependent amino acid decarboxylases.

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Webster, Scott P., Dominic J. Campopiano, Dmitriy Alexeev, Marina Alexeeva, Rory M. Watt, Lindsay Sawyer, and Robert L. Baxter. "Characterisation of 8-amino-7-oxononanoate synthase: A bacterial PLP-dependent, acyl CoA condensing enzyme." Biochemical Society Transactions 26, no. 3 (August 1, 1998): S268. http://dx.doi.org/10.1042/bst026s268.

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Zhang, Hu, Zhao, Huang, Mei, and Mei. "Parallel Strategy Increases the Thermostability and Activity of Glutamate Decarboxylase." Molecules 25, no. 3 (February 6, 2020): 690. http://dx.doi.org/10.3390/molecules25030690.

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Glutamate decarboxylase (GAD; EC 4.1.1.15) is a unique pyridoxal 5-phosphate (PLP)-dependent enzyme that specifically catalyzes the decarboxylation of L-glutamic acid to produce γ-aminobutyric acid (GABA), which exhibits several well-known physiological functions. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, a parallel strategy comprising sequential analysis and free energy calculation was applied to identify critical amino acid sites affecting thermostability of GAD and select proper mutation contributing to improve structure rigidity of the enzyme. Two mutant enzymes, D203E and S325A, with higher thermostability were obtained, and their semi-inactivation temperature (T5015) values were 2.3 °C and 1.4 °C higher than the corresponding value of the wild-type enzyme (WT), respectively. Moreover, the mutant, S325A, exhibited enhanced activity compared to the wild type, with a 1.67-fold increase. The parallel strategy presented in this work proved to be an efficient tool for the reinforcement of protein thermostability.

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Börner, Tim, Carl Grey, and Patrick Adlercreutz. "Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP-dependent enzymes." Biotechnology Journal 11, no. 8 (June 10, 2016): 1025–36. http://dx.doi.org/10.1002/biot.201500587.

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Hasegawa, Takema, Diana Hapsari, and Hitoshi Iwahashi. "RNase H-dependent amplification improves the accuracy of rolling circle amplification combined with loop-mediated isothermal amplification (RCA-LAMP)." PeerJ 9 (July 30, 2021): e11851. http://dx.doi.org/10.7717/peerj.11851.

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The hybrid method upon combining rolling circle amplification and loop-mediated isothermal amplification (RCA-LAMP) was developed to quantify very small amount of different type of RNAs, such as miRNAs. RCA-LAMP can help detect short sequences through padlock probe (PLP) circularization and exhibit powerful DNA amplification. However, one of the factors that determines the detection limit of RCA-LAMP is non-specific amplification. In this study, we improved the accuracy of RCA-LAMP through applying RNase H-dependent PCR (rhPCR) technology. In this method, the non-specific amplification was suppressed by using the rh primer, which is designed through blocking the modification at the 3′end to stop DNA polymerase reaction and replacing the 6th DNA molecule from the end with RNA using RNase H2 enzyme. Traditional RCA-LAMP amplified the non-specific amplicons from linear PLP without a targeting reaction, while RCA-LAMP with rh primer and RNase H2 suppressed the non-specific amplification. Conversely, we identified the risk posed upon conducting PLP cyclization reaction using Splint R ligase in the RNA-targeting step that occurred even in the RNA-negative condition, which is another factor determining the detection limit of RCA-LAMP. Therefore, this study contributes in improving the accuracy of RNA quantification using RCA-LAMP.

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Graham, David E., Stephanie M. Taylor, Rachel Z. Wolf, and Seema C. Namboori. "Convergent evolution of coenzyme M biosynthesis in the Methanosarcinales: cysteate synthase evolved from an ancestral threonine synthase." Biochemical Journal 424, no. 3 (December 10, 2009): 467–78. http://dx.doi.org/10.1042/bj20090999.

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The euryarchaeon Methanosarcina acetivorans has no homologues of the first three enzymes that produce the essential methanogenic coenzyme M (2-mercaptoethanesulfonate) in Methanocaldococcus jannaschii. A single M. acetivorans gene was heterologously expressed to produce a functional sulfopyruvate decarboxylase protein, the fourth canonical enzyme in this biosynthetic pathway. An adjacent gene, at locus MA3297, encodes one of the organism's two threonine synthase homologues. When both paralogues from this organism were expressed in an Escherichia coli threonine synthase mutant, the MA1610 gene complemented the thrC mutation, whereas the MA3297 gene did not. Both PLP (pyridoxal 5′-phosphate)-dependent proteins were heterologously expressed and purified, but only the MA1610 protein catalysed the canonical threonine synthase reaction. The MA3297 protein specifically catalysed a new β-replacement reaction that converted L-phosphoserine and sulfite into L-cysteate and inorganic phosphate. This oxygen-independent mode of sulfonate biosynthesis exploits the facile nucleophilic addition of sulfite to an α,β-unsaturated intermediate (PLP-bound dehydroalanine). An amino acid sequence comparison indicates that cysteate synthase evolved from an ancestral threonine synthase through gene duplication, and the remodelling of active site loop regions by amino acid insertion and substitutions. The cysteate product can be converted into sulfopyruvate by an aspartate aminotransferase enzyme, establishing a new convergent pathway for coenzyme M biosynthesis that appears to function in members of the orders Methanosarcinales and Methanomicrobiales. These differences in coenzyme M biosynthesis afford the opportunity to develop methanogen inhibitors that discriminate between the classes of methanogenic archaea.

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Ho, Thien-Hoang, Kim-Hung Huynh, Diem Quynh Nguyen, Hyunjae Park, Kyoungho Jung, Bookyo Sur, Yeh-Jin Ahn, Sun-Shin Cha, and Lin-Woo Kang. "Catalytic Intermediate Crystal Structures of Cysteine Desulfurase from the Archaeon Thermococcus onnurineus NA1." Archaea 2017 (2017): 1–11. http://dx.doi.org/10.1155/2017/5395293.

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Thermococcus onnurineus NA1 is an anaerobic archaeon usually found in a deep-sea hydrothermal vent area, which can use elemental sulfur (S0) as a terminal electron acceptor for energy. Sulfur, essential to many biomolecules such as sulfur-containing amino acids and cofactors including iron-sulfur cluster, is usually mobilized from cysteine by the pyridoxal 5′-phosphate- (PLP-) dependent enzyme of cysteine desulfurase (CDS). We determined the crystal structures of CDS from Thermococcus onnurineus NA1 (ToCDS), which include native internal aldimine (NAT), gem-diamine (GD) with alanine, internal aldimine structure with existing alanine (IAA), and internal aldimine with persulfide-bound Cys356 (PSF) structures. The catalytic intermediate structures showed the dihedral angle rotation of Schiff-base linkage relative to the PLP pyridine ring. The ToCDS structures were compared with bacterial CDS structures, which will help us to understand the role and catalytic mechanism of ToCDS in the archaeon Thermococcus onnurineus NA1.

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Drake, Eric J., and Andrew M. Gulick. "1.2 Å resolution crystal structure of the periplasmic aminotransferase PvdN fromPseudomonas aeruginosa." Acta Crystallographica Section F Structural Biology Communications 72, no. 5 (April 22, 2016): 403–8. http://dx.doi.org/10.1107/s2053230x16006257.

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The Gram-negative pathogenPseudomonas aeruginosauses a nonribosomal peptide synthetase (NRPS) biosynthetic cluster for the production of a peptide siderophore. In addition to four multimodular NRPS proteins, the biosynthetic pathway also requires several additional enzymes involved in the production of nonproteinogenic amino acids and maturation of the peptide product. Among the proteins that are required for the final steps in pyoverdine synthesis is PvdN, a pyridoxal phosphate-dependent enzyme that catalyzes an uncharacterized step in pyoverdine production. This study reports the high-resolution structure of PvdN bound to a PLP cofactor solved by multi-wavelength anomalous dispersion (MAD). The PvdN model shows high structural homology to type I aspartate aminotransferases and also contains positive density that suggests an uncharacterized external aldimine.

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BERTOLDI, Mariarita, and Carla BORRI VOLTATTORNI. "Reaction of dopa decarboxylase with L-aromatic amino acids under aerobic and anaerobic conditions." Biochemical Journal 352, no. 2 (November 24, 2000): 533–38. http://dx.doi.org/10.1042/bj3520533.

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Analysis of the reaction of dopa decarboxylase (DDC) with L-dopa reveals that loss of decarboxylase activity with time is observed at enzyme concentrations approximately equal to the binding constant, Kd, of the enzyme for pyridoxal 5ƀ-phosphate (PLP). Instead, at enzyme concentrations higher than Kd the course of product formation proceeds linearly until complete consumption of the substrate. Evidence is provided that under both experimental conditions no pyridoxamine 5ƀ-phosphate (PMP) is formed during the reaction and that dissociation of coenzyme occurs at low enzyme concentration, leading to the formation of a PLP-L-dopa Pictet–Spengler cyclic adduct. Taken together, these results indicate that decarboxylation-dependent transamination does not accompany the decarboxylation of L-dopa proposed previously [O'Leary and Baughn (1977) J. Biol. Chem. 252, 7168–7173]. Nevertheless, when the reaction of DDC with L-dopa is studied under anaerobic conditions at an enzyme concentration higher than Kd, we observe that (1) the enzyme is gradually inactivated and inactivation is associated with PMP formation and (2) the initial velocity of decarboxylation is approximately half of that in the presence of O2. Similar behaviour is observed by comparing the reaction with L-5-hydroxytryptophan occurring in aerobiosis or in anaerobiosis. Therefore the reaction of DDC with L-aromatic amino acids seems to be under O2 control. In contrast, the reactivity of the enzyme with D-aromatic amino acids does not change in the presence or absence of O2. These and other results, together with previous results on the effect exerted by O2 on reaction specificity of DDC towards aromatic amines [Bertoldi, Frigeri, Paci and Borri Voltattorni (1999) J. Biol. Chem. 274, 5514–5521], suggest a productive effect of O2 on an intermediate complex of the reaction of the enzyme with L-aromatic amino acids or aromatic amines.

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Fujino, A., T. Ose, M. Yao, M. Honma, and I. Tanaka. "Catalytic activity analysis of PLP dependent enzyme PH0054 from hyperthermophilic archaebacteria P.horikoshii OT3 by X-ray crystallography." Seibutsu Butsuri 41, supplement (2001): S100. http://dx.doi.org/10.2142/biophys.41.s100_4.

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Lambrecht, Jennifer A., Jeffrey M. Flynn, and Diana M. Downs. "Conserved YjgF Protein Family Deaminates Reactive Enamine/Imine Intermediates of Pyridoxal 5′-Phosphate (PLP)-dependent Enzyme Reactions." Journal of Biological Chemistry 287, no. 5 (November 17, 2011): 3454–61. http://dx.doi.org/10.1074/jbc.m111.304477.

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Murphy, Cormac D., David O'Hagan, and Christoph Schaffrath. "Identification of a PLP-Dependent Threonine Transaldolase: A Novel Enzyme Involved in 4-Fluorothreonine Biosynthesis inStreptomyces cattleya." Angewandte Chemie 113, no. 23 (December 3, 2001): 4611–13. http://dx.doi.org/10.1002/1521-3757(20011203)113:23<4611::aid-ange4611>3.0.co;2-u.

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Nguyen, Diem-Quynh, Ho-Phuong-Thuy Ngo, Yeh-Jin Ahn, Sang Hee Lee, and Lin-Woo Kang. "Expression, crystallization and preliminary X-ray crystallographic analysis of cystathionine β-lyase fromAcinetobacter baumanniiOXA-23." Acta Crystallographica Section F Structural Biology Communications 70, no. 10 (September 25, 2014): 1368–71. http://dx.doi.org/10.1107/s2053230x14017981.

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Multidrug-resistantAcinetobacter baumannii(Ab) has emerged as a leading nosocomial pathogen because of its resistance to most currently available antibiotics. Cystathionine β-lyase (CBL), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, catalyzes the second step in the transsulfuration pathway, which is essential for the metabolic interconversion of the sulfur-containing amino acids homocysteine and methionine. The enzymes of the transsulfuration pathway are considered to be attractive drug targets owing to their specificity to microbes and plants. As a potential target for the development of novel antibacterial drugs, the AbCBL protein was expressed, purified and crystallized. An AbCBL crystal diffracted to 1.57 Å resolution and belonged to the trigonal space groupP3112, with unit-cell parametersa=b= 102.9,c= 136.5 Å. The asymmetric unit contained two monomers, with a correspondingVMof 2.3 Å3 Da−1and a solvent content of 46.9%.

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Marienhagen, Jan, Nicole Kennerknecht, Hermann Sahm, and Lothar Eggeling. "Functional Analysis of All Aminotransferase Proteins Inferred from the Genome Sequence of Corynebacterium glutamicum." Journal of Bacteriology 187, no. 22 (November 15, 2005): 7639–46. http://dx.doi.org/10.1128/jb.187.22.7639-7646.2005.

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ABSTRACT Twenty putative aminotransferase (AT) proteins of Corynebacterium glutamicum, or rather pyridoxal-5′-phosphate (PLP)-dependent enzymes, were isolated and assayed among others with l-glutamate, l-aspartate, and l-alanine as amino donors and a number of 2-oxo-acids as amino acceptors. One outstanding AT identified is AlaT, which has a broad amino donor specificity utilizing (in the order of preference) l-glutamate > 2-aminobutyrate > l-aspartate with pyruvate as acceptor. Another AT is AvtA, which utilizes l-alanine to aminate 2-oxo-isovalerate, the l-valine precursor, and 2-oxo-butyrate. A second AT active with the l-valine precursor and that of the other two branched-chain amino acids, too, is IlvE, and both enzyme activities overlap partially in vivo, as demonstrated by the analysis of deletion mutants. Also identified was AroT, the aromatic AT, and this and IlvE were shown to have comparable activities with phenylpyruvate, thus demonstrating the relevance of both ATs for l-phenylalanine synthesis. We also assessed the activity of two PLP-containing cysteine desulfurases, supplying a persulfide intermediate. One of them is SufS, which assists in the sulfur transfer pathway for the Fe-S cluster assembly. Together with the identification of further ATs and the additional analysis of deletion mutants, this results in an overview of the ATs within an organism that may not have been achieved thus far.

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Journal articles on the topic 'PLP-dependent enzyme'

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