Journal articles: 'NADP-bound'

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GAETANI, Gian F., Anna M. FERRARIS, Paola SANNA, and Henry N. KIRKMAN. "A novel NADPH:(bound) NADP+ reductase and NADH:(bound) NADP+ transhydrogenase function in bovine liver catalase." Biochemical Journal 385, no. 3 (January 24, 2005): 763–68. http://dx.doi.org/10.1042/bj20041495.

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Many catalases have the shared property of containing bound NADPH and being susceptible to inactivation by their own substrate, H2O2. The presence of additional (unbound) NADPH effectively prevents bovine liver and human erythrocytic catalase from becoming compound II, the reversibly inactivated state of catalase, and NADP+ is known to be generated in the process. The function of the bound NADPH, which is tightly bound in bovine liver catalase, has been unknown. The present study with bovine liver catalase and [14C]NADPH and [14C]NADH revealed that unbound NADPH or NADH are substrates for an internal reductase and transhydrogenase reaction respectively; the unbound NADPH or NADH cause tightly bound NADP+ to become NADPH without becoming tightly bound themselves. This and other results provide insight into the function of tightly bound NADPH.

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Kondo, Hisanori, and Midori Murakami. "Crystal Structures of the Putative Isocitrate Dehydrogenase from Sulfolobus tokodaii Strain 7 in the Apo and NADP+-Bound Forms." Archaea 2018 (December 19, 2018): 1–9. http://dx.doi.org/10.1155/2018/7571984.

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Isocitrate dehydrogenase is a catabolic enzyme that acts during the third step of the tricarboxylic acid cycle. The hypothetical protein ST2166 from the archaeon Sulfolobus tokodaii was isolated and crystallized. It shares high primary structure homology with prokaryotic NADP+-dependent IDHs, suggesting that these enzymes share a common enzymatic mechanism. The crystal structure of ST2166 was determined at 2.0 Å resolution in the apo form, and then the structure of the crystal soaked with NADP+ was also determined at 2.4 Å resolution, which contained NADP+ bound at the putative active site. Comparisons between the structures of apo and NADP+-bound forms and NADP-IDHs from other prokaryotes suggest that prokaryotic NADP-IDHs recognize their cofactors using conserved Lys335, Tyr336, and Arg386 in ST2166 at the opening cleft before the domain closure.

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Verma, Rajni, Jonathan M. Ellis, and Katie R. Mitchell-Koch. "Dynamic Preference for NADP/H Cofactor Binding/Release in E. coli YqhD Oxidoreductase." Molecules 26, no. 2 (January 7, 2021): 270. http://dx.doi.org/10.3390/molecules26020270.

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YqhD, an E. coli alcohol/aldehyde oxidoreductase, is an enzyme able to produce valuable bio-renewable fuels and fine chemicals from a broad range of starting materials. Herein, we report the first computational solution-phase structure-dynamics analysis of YqhD, shedding light on the effect of oxidized and reduced NADP/H cofactor binding on the conformational dynamics of the biocatalyst using molecular dynamics (MD) simulations. The cofactor oxidation states mainly influence the interdomain cleft region conformations of the YqhD monomers, involved in intricate cofactor binding and release. The ensemble of NADPH-bound monomers has a narrower average interdomain space resulting in more hydrogen bonds and rigid cofactor binding. NADP-bound YqhD fluctuates between open and closed conformations, while it was observed that NADPH-bound YqhD had slower opening/closing dynamics of the cofactor-binding cleft. In the light of enzyme kinetics and structural data, simulation findings have led us to postulate that the frequently sampled open conformation of the cofactor binding cleft with NADP leads to the more facile release of NADP while increased closed conformation sampling during NADPH binding enhances cofactor binding affinity and the aldehyde reductase activity of the enzyme.

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Verma, Rajni, Jonathan M. Ellis, and Katie R. Mitchell-Koch. "Dynamic Preference for NADP/H Cofactor Binding/Release in E. coli YqhD Oxidoreductase." Molecules 26, no. 2 (January 7, 2021): 270. http://dx.doi.org/10.3390/molecules26020270.

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YqhD, an E. coli alcohol/aldehyde oxidoreductase, is an enzyme able to produce valuable bio-renewable fuels and fine chemicals from a broad range of starting materials. Herein, we report the first computational solution-phase structure-dynamics analysis of YqhD, shedding light on the effect of oxidized and reduced NADP/H cofactor binding on the conformational dynamics of the biocatalyst using molecular dynamics (MD) simulations. The cofactor oxidation states mainly influence the interdomain cleft region conformations of the YqhD monomers, involved in intricate cofactor binding and release. The ensemble of NADPH-bound monomers has a narrower average interdomain space resulting in more hydrogen bonds and rigid cofactor binding. NADP-bound YqhD fluctuates between open and closed conformations, while it was observed that NADPH-bound YqhD had slower opening/closing dynamics of the cofactor-binding cleft. In the light of enzyme kinetics and structural data, simulation findings have led us to postulate that the frequently sampled open conformation of the cofactor binding cleft with NADP leads to the more facile release of NADP while increased closed conformation sampling during NADPH binding enhances cofactor binding affinity and the aldehyde reductase activity of the enzyme.

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Lobley, Carina M. C., Alessio Ciulli, Heather M. Whitney, Glyn Williams, Alison G. Smith, Chris Abell, and Tom L. Blundell. "The Crystal Structure ofEscherichia coliKetopantoate Reductase with NADP+Bound†,‡." Biochemistry 44, no. 25 (June 2005): 8930–39. http://dx.doi.org/10.1021/bi0502036.

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OKUDA, Keiko, Itaru URABE, and Hirosuke OKADA. "Synthesis of poly(ethylene glycol)-bound NADP by selective modification at the 6-amino group of NADP." European Journal of Biochemistry 151, no. 1 (August 1985): 33–38. http://dx.doi.org/10.1111/j.1432-1033.1985.tb09065.x.

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Padyana, Anil K., and Stephen K. Burley. "Crystal Structure of Shikimate 5-Dehydrogenase (SDH) Bound to NADP." Structure 11, no. 8 (August 2003): 1005–13. http://dx.doi.org/10.1016/s0969-2126(03)00159-x.

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8

Brito, Rui M. M., Frederick B. Rudolph, and Paul R. Rosevear. "Conformation of NADP+ bound to a type II dihydrofolate reductase." Biochemistry 30, no. 6 (February 12, 1991): 1461–69. http://dx.doi.org/10.1021/bi00220a003.

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9

Anderlund, Mikael, Torben L. Nissen, Jens Nielsen, John Villadsen, Jan Rydström, Bärbel Hahn-Hägerdal, and Morten C. Kielland-Brandt. "Expression of the Escherichia coli pntA andpntB Genes, Encoding Nicotinamide Nucleotide Transhydrogenase, in Saccharomyces cerevisiae and Its Effect on Product Formation during Anaerobic Glucose Fermentation." Applied and Environmental Microbiology 65, no. 6 (June 1, 1999): 2333–40. http://dx.doi.org/10.1128/aem.65.6.2333-2340.1999.

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ABSTRACT We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinantSaccharomyces cerevisiae expressing the membrane-bound transhydrogenase from Escherichia coli. Our objective was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD+ in S. cerevisiaeand thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD+ by NADPH and by NADH in the presence of NADP+, which demonstrated that an active enzyme was present. Unlike the situation in E. coli, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD+, NADPH, and NADP+were measured in cells expressing transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD+.

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Liu, Si-Qi, Hongjun Jin, Albert Zacarias, Sanjay Srivastava, and Aruni Bhatnagar. "Binding of Pyridine Nucleotide Coenzymes to the β-Subunit of the Voltage-sensitive K+Channel." Journal of Biological Chemistry 276, no. 15 (January 17, 2001): 11812–20. http://dx.doi.org/10.1074/jbc.m008259200.

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The β-subunit of the voltage-sensitive K+(Kv) channels belongs to the aldo-keto reductase superfamily, and the crystal structure of Kvβ2 shows NADP bound in its active site. Here we report that Kvβ2 displays a high affinity for NADPH (Kd= 0.1 μm) and NADP+(Kd= 0.3 μm), as determined by fluorometric titrations of the recombinant protein. The Kvβ2 also bound NAD(H) but with 10-fold lower affinity. The site-directed mutants R264E and N333W did not bind NADPH, whereas, theKdNADPHof Q214R was 10-fold greater than the wild-type protein. TheKdNADPHwas unaffected by the R189M, W243Y, W243A, or Y255F mutation. The tetrameric structure of the wild-type protein was retained by the R264E mutant, indicating that NADPH binding is not a prerequisite for multimer formation. A C248S mutation caused a 5-fold decrease inKdNADPH, shifted the pKaofKdNADPHfrom 6.9 to 7.4, and decreased the ionic strength dependence of NADPH binding. These results indicate that Arg-264 and Asn-333 are critical for coenzyme binding, which is regulated in part by Cys-248. The binding of both NADP(H) and NAD(H) to the protein suggests that several types of Kvβ2-nucleotide complexes may be formedin vivo.

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11

Leisch, Hannes, Rong Shi, Stephan Grosse, Krista Morley, Hélène Bergeron, Miroslaw Cygler, Hiroaki Iwaki, Yoshie Hasegawa, and Peter C. K. Lau. "Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ3-4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453." Applied and Environmental Microbiology 78, no. 7 (January 20, 2012): 2200–2212. http://dx.doi.org/10.1128/aem.07694-11.

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ABSTRACTA dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor byPseudomonas putidaATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) inEscherichia coliand determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP+at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP+. A comparison of several crystal forms of OTEMO bound to FAD and NADP+revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (kcat/Km) favors 2-n-hexyl cyclopentanone (4.3 × 105M−1s−1) as a substrate, although its affinity (Km= 32 μM) was lower than that of the CoA-activated substrate (Km= 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.

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Vinkovic, M., G. Dunn, G. E. Wood, J. Husain, S. P. Wood, and R. Gill. "Cleavage of nicotinamide adenine dinucleotide by the ribosome-inactivating protein fromMomordica charantia." Acta Crystallographica Section F Structural Biology Communications 71, no. 9 (August 25, 2015): 1152–55. http://dx.doi.org/10.1107/s2053230x15013540.

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The interaction of momordin, a type 1 ribosome-inactivating protein fromMomordica charantia, with NADP+and NADPH has been investigated by X-ray diffraction analysis of complexes generated by co-crystallization and crystal soaking. It is known that the proteins of this family readily cleave the adenine–ribose bond of adenosine and related nucleotides in the crystal, leaving the product, adenine, bound to the enzyme active site. Surprisingly, the nicotinamide–ribose bond of oxidized NADP+is cleaved, leaving nicotinamide bound in the active site in the same position but in a slightly different orientation to that of the five-membered ring of adenine. No binding or cleavage of NADPH was observed at pH 7.4 in these experiments. These observations are in accord with current views of the enzyme mechanism and may contribute to ongoing searches for effective inhibitors.

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13

Warkentin, E. "Structures of F420H2:NADP+ oxidoreductase with and without its substrates bound." EMBO Journal 20, no. 23 (December 3, 2001): 6561–69. http://dx.doi.org/10.1093/emboj/20.23.6561.

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Liu, Xin-Xin, Wei-bing Liu, and Bang-Ce Ye. "Regulation of a Protein Acetyltransferase in Myxococcus xanthus by the Coenzyme NADP+." Journal of Bacteriology 198, no. 4 (November 23, 2015): 623–32. http://dx.doi.org/10.1128/jb.00661-15.

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ABSTRACTNADP+is a vital cofactor involved in a wide variety of activities, such as redox potential and cell death. Here, we show that NADP+negatively regulates an acetyltransferase fromMyxococcus xanthus, Mxan_3215 (MxKat), at physiologic concentrations.MxKat possesses an NAD(P)-binding domain fused to the Gcn5-typeN-acetyltransferase (GNAT) domain. We used isothermal titration calorimetry (ITC) and a coupled enzyme assay to show that NADP+bound toMxKat and that the binding had strong effects on enzyme activity. The Gly11 residue ofMxKat was confirmed to play an important role in NADP+binding using site-directed mutagenesis and circular dichroism spectrometry. In addition, using mass spectrometry, site-directed mutagenesis, and a coupling enzymatic assay, we demonstrated thatMxKat acetylates acetyl coenzyme A (acetyl-CoA) synthetase (Mxan_2570) at Lys622 in response to changes in NADP+concentration. Collectively, our results uncovered a mechanism of protein acetyltransferase regulation by the coenzyme NADP+at physiological concentrations, suggesting a novel signaling pathway for the regulation of cellular protein acetylation.IMPORTANCEMicroorganisms have developed various protein posttranslational modifications (PTMs), which enable cells to respond quickly to changes in the intracellular and extracellular milieus. This work provides the first biochemical characterization of a protein acetyltransferase (MxKat) that contains a fusion between a GNAT domain and NADP+-binding domain with Rossmann folds, and it demonstrates a novel signaling pathway for regulating cellular protein acetylation inM. xanthus. We found that NADP+specifically binds to the Rossmann fold ofMxKat and negatively regulates its acetyltransferase activity. This finding provides novel insight for connecting cellular metabolic status (NADP+metabolism) with levels of protein acetylation, and it extends our understanding of the regulatory mechanisms underlying PTMs.

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Pfoh, Roland, Emil F. Pai, and Vivian Saridakis. "Nicotinamide mononucleotide adenylyltransferase displays alternate binding modes for nicotinamide nucleotides." Acta Crystallographica Section D Biological Crystallography 71, no. 10 (September 26, 2015): 2032–39. http://dx.doi.org/10.1107/s1399004715015497.

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Nicotinamide mononucleotide adenylyltransferase (NMNAT) catalyzes the biosynthesis of NAD+and NaAD+. The crystal structure of NMNAT fromMethanobacterium thermoautotrophicumcomplexed with NAD+and SO42−revealed the active-site residues involved in binding and catalysis. Site-directed mutagenesis was used to further characterize the roles played by several of these residues. Arg11 and Arg136 were implicated in binding the phosphate groups of the ATP substrate. Both of these residues were mutated to lysine individually. Arg47 does not interact with either NMN or ATP substrates directly, but was deemed to play a role in binding as it is proximal to Arg11 and Arg136. Arg47 was mutated to lysine and glutamic acid. Surprisingly, when expressed inEscherichia coliall of these NMNAT mutants trapped a molecule of NADP+in their active sites. This NADP+was bound in a conformation that was quite different from that displayed by NAD+in the native enzyme complex. When NADP+was co-crystallized with wild-type NMNAT, the same structural arrangement was observed. These studies revealed a different conformation of NADP+in the active site of NMNAT, indicating plasticity of the active site.

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KNIGHT, Kirsty, and Nigel S. SCRUTTON. "Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase." Biochemical Journal 367, no. 1 (October 1, 2002): 19–30. http://dx.doi.org/10.1042/bj20020667.

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The reduction by NADPH of the FAD and FMN redox centres in the isolated flavin reductase domain of calmodulin-bound rat neuronal nitric oxide synthase (nNOS) has been studied by anaerobic stopped-flow spectroscopy using absorption and fluorescence detection. We show by global analysis of time-dependent photodiode array spectra, single wavelength absorption and NADPH fluorescence studies, that at least four resolvable steps are observed in stopped-flow studies with NADPH and that flavin reduction is reversible. The first reductive step represents the rapid formation of an equilibrium between an NADPH-enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP+. The second and third steps represent further reduction of the enzyme flavins and NADP+ release. The fourth step is attributed to the slow accumulation of an enzyme species that is inferred not to be relevant catalytically in steady-state reactions. Stopped-flow flavin fluorescence studies indicate the presence of slow kinetic phases, the timescales of which correspond to the slow phase observed in absorption and NADPH fluorescence transients. By analogy with stopped-flow studies of cytochrome P450 reductase, we attribute these slow fluorescence and absorption changes to enzyme disproportionation and/or conformational change. Unlike for the functionally related cytochrome P450 reductase, transfer of the first hydride equivalent from NADPH to nNOS reductase does not generate the flavin di-semiquinoid state. This indicates that internal electron transfer is relatively slow and is probably gated by NADP+ release. Release of calmodulin from the nNOS reductase does not affect the kinetics of inter-flavin electron transfer under stopped-flow conditions, although the observed rate of formation of the equilibrium between the NADPH-oxidized enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP+ is modestly slower in calmodulin-depleted enzyme. Our studies indicate the need for significant re-interpretation of published kinetic data for electron transfer in the reductase domain of neuronal nitric oxide synthase.

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Wang, Rui, Jiaquan Wu, David Kin Jin, Yali Chen, Zhijia Lv, Qian Chen, Qiwei Miao, Xiaoyu Huo, and Feng Wang. "Structure of NADP+-bound 7β-hydroxysteroid dehydrogenase reveals two cofactor-binding modes." Acta Crystallographica Section F Structural Biology Communications 73, no. 5 (April 26, 2017): 246–52. http://dx.doi.org/10.1107/s2053230x17004460.

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In mammals, bile acids/salts and their glycine and taurine conjugates are effectively recycled through enterohepatic circulation. 7β-Hydroxysteroid dehydrogenases (7β-HSDHs; EC 1.1.1.201), including that from the intestinal microbeCollinsella aerofaciens, catalyse the NADPH-dependent reversible oxidation of secondary bile-acid products to avoid potential toxicity. Here, the first structure of NADP+bound to dimeric 7β-HSDH is presented. In one active site, NADP+adopts a conventional binding mode similar to that displayed in related enzyme structures. However, in the other active site a unique binding mode is observed in which the orientation of the nicotinamide is different. Since 7β-HSDH has become an attractive target owing to the wide and important pharmaceutical use of its product ursodeoxycholic acid, this work provides a more detailed template to support rational protein engineering to improve the enzymatic activities of this useful biocatalyst, further improving the yield of ursodeoxycholic acid and its other applications.

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MATSUURA, Kazuya, Yoshiyuki TAMADA, Kumiko SATO, Harunori IWASA, Gunpei MIWA, Yoshihiro DEYASHIKI, and Akira HARA. "Involvement of two basic residues (Lys-270 and Arg-276) of human liver 3α-hydroxysteroid dehydrogenase in NADP(H) binding and activation by sulphobromophthalein: site-directed mutagenesis and kinetic analysis." Biochemical Journal 322, no. 1 (February 15, 1997): 89–93. http://dx.doi.org/10.1042/bj3220089.

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A human liver 3α-hydroxysteroid dehydrogenase isoenzyme, a member of the aldoŐketo reductase family, shows a marked preference for NADP(H) over NAD(H), and is activated by sulphobromophthalein, which increases the Km values for both NADP(H) and substrates. Here we report kinetic alterations in binding of the coenzymes and the activator to the enzyme caused by site-directed mutagenesis of Lys-270 and Arg-276, which are strictly conserved among the aldoŐketo reductase family of enzymes. The mutated enzymes, K270M and R276M, showed increases in the Km for NADP+ of 22- and 290-fold respectively; the Km for alcohol substrate and the kcat of the NADP+-linked reaction were also elevated, by 9- and 5-fold respectively. No kinetic constant of the NAD+-linked reaction was altered by more than 3-fold. Calculation of the free-energy changes showed that the 2ƀ-phosphate group of NADP+ contributes 16.3 kJ/mol (3.9 kcal/mol) of binding energy to its interaction with the wild-type enzyme, and the mutagenesis to K270M and R276M destabilized the binding energy of NADP+ by 6.3 and 13.0 kJ/mol (1.5 and 3.1 kcal/mol) respectively. In addition, the mutations attenuated enzyme activation by sulphobromophthalein, which bound to the mutant enzymes as an inhibitor. The inhibition for the R276M mutant was competitive with respect to NADP+ and non-competitive with respect to the substrate, whereas that for the K270M mutant was mixed-type, showing activation at coenzyme concentrations greater than 20ȕKm. These results suggest that the two basic residues in the 3α-hydroxysteroid dehydrogenase isoenzyme play crucial roles in binding both the negatively charged 2ƀ-phosphate group of NADP+ and the sulphonic groups of sulphobromophthalein.

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Kim, Gyuhee, Donghyuk Shin, Sumin Lee, Jaesook Yun, and Sangho Lee. "Crystal Structure of IlvC, a Ketol-Acid Reductoisomerase, from Streptococcus Pneumoniae." Crystals 9, no. 11 (October 24, 2019): 551. http://dx.doi.org/10.3390/cryst9110551.

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Biosynthesis of branched-chain amino acids (BCAAs), including isoleucine, leucine and valine, is required for survival and virulence of a bacterial pathogen such as Streptococcus pneumoniae. IlvC, a ketol-acid reductoisomerase (E.C. 1.1.1.86) with NADP(H) and Mg2+ as cofactors from the pathogenic Streptococcus pneumoniae (SpIlvC), catalyzes the second step in the BCAA biosynthetic pathway. To elucidate the structural basis for the IlvC-mediated reaction, we determined the crystal structure of SpIlvC at 1.69 Å resolution. The crystal structure of SpIlvC contains an asymmetric dimer in which one subunit is in apo-form and the other in NADP(H) and Mg2+-bound form. Crystallographic analysis combined with an activity assay and small-angle X-ray scattering suggested that SpIlvC retains dimeric arrangement in solution and that D83 in the NADP(H) binding site and E195 in the Mg2+ binding site are the most critical in the catalytic activity of SpIlvC. Crystal structures of SpIlvC mutants (R49E, D83G, D191G and E195S) revealed local conformational changes only in the NADP(H) binding site. Taken together, our results establish the molecular mechanism for understanding functions of SpIlvC in pneumococcal growth and virulence.

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Argyrou, Argyrides, Matthew W. Vetting, and John S. Blanchard. "Characterization of a New Member of the Flavoprotein Disulfide Reductase Family of Enzymes fromMycobacterium tuberculosis." Journal of Biological Chemistry 279, no. 50 (September 29, 2004): 52694–702. http://dx.doi.org/10.1074/jbc.m410704200.

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ThelpdA(Rv3303c) gene fromMycobacterium tuberculosisencoding a new member of the flavoprotein disulfide reductases was expressed inEscherichia coli, and the recombinant LpdA protein was purified to homogeneity. LpdA is a homotetramer and co-purifies with one molecule of tightly but noncovalently bound FAD and NADP+per monomer. Although annotated as a probable lipoamide dehydrogenase inM. tuberculosis, LpdA cannot catalyze reduction of lipoyl substrates, because it lacks one of two cysteine residues involved in dithiol-disulfide interchange with lipoyl substrates and a His-Glu pair involved in general acid catalysis. The crystal structure of LpdA was solved by multiple isomorphous replacement with anomalous scattering, which confirmed the absence of these catalytic residues from the active site. Although LpdA cannot catalyze reduction of disulfide-bonded substrates, it catalyzes the NAD(P)H-dependent reduction of alternative electron acceptors such as 2,6-dimethyl-1,4-benzoquinone and 5-hydroxy-1,4-naphthaquinone. Significant primary deuterium kinetic isotope effects were observed with [4S-2H]NADH establishing that the enzyme promotes transfer of the C4-proShydride of NADH. The absence of an isotope effect with [4S-2H]NADPH, the lowKmvalue of 0.5 μmfor NADPH, and the potent inhibition of the NADH-dependent reduction of 2,6-dimethyl-1,4-benzoquinone by NADP+(Ki∼ 6 nm) and 2′-phospho-ADP-ribose (Ki∼ 800 nm), demonstrate the high affinity of LpdA for 2′-phosphorylated nucleotides and that the physiological substrate/product pair is NADPH/NADP+rather than NADH/NAD+. Modeling of NADP+in the active site revealed that LpdA achieves the high specificity for NADP+through interactions involving the 2′-phosphate of NADP+and amino acid residues that are different from those in glutathione reductase.

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Yokota, A., S. Haga, and S. Kitaoka. "Purification and some properties of glyoxylate reductase (NADP+) and its functional location in mitochondria in Euglena gracilis z." Biochemical Journal 227, no. 1 (April 1, 1985): 211–16. http://dx.doi.org/10.1042/bj2270211.

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Euglena mitochondria contain both glyoxylate reductase (NADP+) and glycollate dehydrogenase to constitute the glycollate-glyoxylate cycle [Yokota & Kitaoka (1979) Biochem. J. 184, 189-192]. Euglena glyoxylate reductase (NADP+) was purified and its submitochondrial location was determined in order to elucidate the cycle. The purified glyoxylate reductase was homogeneous on polyacrylamide-gel electrophoresis. Difference spectra of the purified enzyme revealed that the enzyme was a flavin enzyme. The Mr of the enzyme was 82 000. The enzyme was specific for NADPH, with an apparent Km of 3.9 microM, and for glyoxylate, with an apparent Km of 45 microM. It was 30% as active with oxaloacetate as with glyoxylate. NADH and hydroxypyruvate did not support the activity at all. The optimum pH was 6.45. Submitochondrial fractionation of purified mitochondria showed that the enzyme was located in the intermembrane space and loosely bound to the outer surface of the inner membrane. These properties and the submitochondrial localization of NADPH-glyoxylate reductase facilitate the operation of the glycollate-glyoxylate cycle in combination with glycollate dehydrogenase, which is tightly bound to the inner membrane of Euglena mitochondria.

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Küssau, Tanja, Marion Flipo, Niel Van Wyk, Albertus Viljoen, Vincent Olieric, Laurent Kremer, and Mickaël Blaise. "Structural rearrangements occurring upon cofactor binding in the Mycobacterium smegmatis β-ketoacyl-acyl carrier protein reductase MabA." Acta Crystallographica Section D Structural Biology 74, no. 5 (April 24, 2018): 383–93. http://dx.doi.org/10.1107/s2059798318002917.

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In mycobacteria, the ketoacyl-acyl carrier protein (ACP) reductase MabA (designated FabG in other bacteria) catalyzes the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products. This first reductive step in the fatty-acid biosynthesis elongation cycle is essential for bacteria, which makes MabA/FabG an interesting drug target. To date, however, very few molecules targeting FabG have been discovered and MabA remains the only enzyme of the mycobacterial type II fatty-acid synthase that lacks specific inhibitors. Despite the existence of several MabA/FabG crystal structures, the structural rearrangement that occurs upon cofactor binding is still not fully understood. Therefore, unlocking this knowledge gap could help in the design of new inhibitors. Here, high-resolution crystal structures of MabA from Mycobacterium smegmatis in its apo, NADP+-bound and NADPH-bound forms are reported. Comparison of these crystal structures reveals the structural reorganization of the lid region covering the active site of the enzyme. The crystal structure of the apo form revealed numerous residues that trigger steric hindrance to the binding of NADPH and substrate. Upon NADPH binding, these residues are pushed away from the active site, allowing the enzyme to adopt an open conformation. The transition from an NADPH-bound to an NADP+-bound form is likely to facilitate release of the product. These results may be useful for subsequent rational drug design and/or for in silico drug-screening approaches targeting MabA/FabG.

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Saliola, Michele, Angela Tramonti, Claudio Lanini, Samantha Cialfi, Daniela De Biase, and Claudio Falcone. "Intracellular NADPH Levels Affect the Oligomeric State of the Glucose 6-Phosphate Dehydrogenase." Eukaryotic Cell 11, no. 12 (October 12, 2012): 1503–11. http://dx.doi.org/10.1128/ec.00211-12.

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ABSTRACTIn the yeastKluyveromyces lactis, glucose 6-phosphate dehydrogenase (G6PDH) is detected as two differently migrating forms on native polyacrylamide gels. The pivotal metabolic role of G6PDH inK. lactisled us to investigate the mechanism controlling the two activities in respiratory and fermentative mutant strains. An extensive analysis of these mutants showed that the NAD+(H)/NADP+(H)-dependent cytosolic alcohol (ADH) and aldehyde (ALD) dehydrogenase balance affects the expression of the G6PDH activity pattern. Under fermentative/ethanol growth conditions, the concomitant activation of ADH and ALD activities led to cytosolic accumulation of NADPH, triggering an alteration in the oligomeric state of the G6PDH caused by displacement/release of the structural NADP+bound to each subunit of the enzyme. The new oligomeric G6PDH form with faster-migrating properties increases as a consequence of intracellular redox unbalance/NADPH accumulation, which inhibits G6PDH activityin vivo. The appearance of a new G6PDH-specific activity band, following incubation ofSaccharomyces cerevisiaeand human cellular extracts with NADP+, also suggests that a regulatory mechanism of this activity through NADPH accumulation is highly conserved among eukaryotes.

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Lintala, Minna, Natalie Schuck, Ina Thormählen, Andreas Jungfer, Katrin L. Weber, Andreas P. M. Weber, Peter Geigenberger, Jürgen Soll, Bettina Bölter, and Paula Mulo. "Arabidopsis tic62 trol Mutant Lacking Thylakoid-Bound Ferredoxin–NADP+ Oxidoreductase Shows Distinct Metabolic Phenotype." Molecular Plant 7, no. 1 (January 2014): 45–57. http://dx.doi.org/10.1093/mp/sst129.

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Ciulli, Alessio, Dimitri Y. Chirgadze, Alison G. Smith, Tom L. Blundell, and Chris Abell. "Crystal Structure ofEscherichia coliKetopantoate Reductase in a Ternary Complex with NADP+and Pantoate Bound." Journal of Biological Chemistry 282, no. 11 (January 16, 2007): 8487–97. http://dx.doi.org/10.1074/jbc.m611171200.

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Grimshaw, Charles E. "Enantiospecific change in products for aldose reductase-mediated reaction of glyceraldehyde with bound NADP+." Biochemical and Biophysical Research Communications 175, no. 3 (March 1991): 943–48. http://dx.doi.org/10.1016/0006-291x(91)91656-w.

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AHVAZI, Bijan, Rene COULOMBE, Marc DELARGE, Masoud VEDADI, Lei ZHANG, Edward MEIGHEN, and Alice VRIELINK. "Crystal structure of the NADP+-dependent aldehyde dehydrogenase from Vibrio harveyi: structural implications for cofactor specificity and affinity." Biochemical Journal 349, no. 3 (July 25, 2000): 853–61. http://dx.doi.org/10.1042/bj3490853.

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Aldehyde dehydrogenase from the bioluminescent bacterium, Vibrio harveyi, catalyses the oxidation of long-chain aliphatic aldehydes to acids. The enzyme is unique compared with other forms of aldehyde dehydrogenase in that it exhibits a very high specificity and affinity for the cofactor NADP+. Structural studies of this enzyme and comparisons with other forms of aldehyde dehydrogenase provide the basis for understanding the molecular features that dictate these unique properties and will enhance our understanding of the mechanism of catalysis for this class of enzyme. The X-ray structure of aldehyde dehydrogenase from V. harveyi has been solved to 2.5-Å resolution as a partial complex with the cofactor NADP+ and to 2.1-Å resolution as a fully bound ‘holo’complex. The cofactor preference exhibited by different forms of the enzyme is predominantly determined by the electrostatic environment surrounding the 2´-hydroxy or the 2´-phosphate groups of the adenosine ribose moiety of NAD+ or NADP+, respectively. In the NADP+-dependent structures the presence of a threonine and a lysine contribute to the cofactor specificity. In the V. harveyi enzyme an arginine residue (Arg-210) contributes to the high cofactor affinity through a pi stacking interaction with the adenine ring system of the cofactor. Further differences between the V. harveyi enzyme and other aldehyde dehydrogenases are seen in the active site, in particular a histidine residue which is structurally conserved with phosphorylating glyceraldehyde-3-phosphate dehydrogenase. This may suggest an alternative mechanism for activation of the reactive cysteine residue for nucleophilic attack.

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Tsai, C. Stan, and D. J. Senior. "Dual coenzyme activities of high-Km aldehyde dehydrogenase from rat liver mitochondria." Biochemistry and Cell Biology 68, no. 4 (April 1, 1990): 751–57. http://dx.doi.org/10.1139/o90-108.

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Various kinetic approaches were carried out to investigate kinetic attributes for the dual coenzyme activities of mitochondrial aldehyde dehydrogenase from rat liver. The enzyme catalyses NAD+- and NADP+-dependent oxidations of ethanal by an ordered bi-bi mechanism with NAD(P)+ as the first reactant bound and NAD(P)H as the last product released. The two coenzymes presumably interact with the kinetically identical site. NAD+ forms the dynamic binary complex with the enzyme, while the enzyme-NAD(P)H complex formation is associated with conformation change(s). A stopped-flow burst of NAD(P)H formation, followed by a slower steady-state turnover, suggests that either the deacylation or the release of NAD(P)H is rate limiting. Although NADP+ is reduced by a faster burst rate, NAD+ is slightly favored as the coenzyme by virtue of its marginally faster turnover rate.Key words: aldehyde dehydrogenase, coenzyme preference.

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Takei, H., K. Nakashima, O. Adachi, E. Shinagawa, and M. Ameyama. "Enzymatic determination of serum ethanol with membrane-bound dehydrogenase." Clinical Chemistry 31, no. 12 (December 1, 1985): 1985–87. http://dx.doi.org/10.1093/clinchem/31.12.1985.

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Abstract In this enzymatic method for detecting ethanol in blood by use of membrane-bound microbial alcohol dehydrogenase (no EC no. assigned), the enzyme catalyzes the reaction irreversibly and the rate of oxidation can be monitored by spectrophotometry of the reduction of the indicator dye. No pyridine nucleotides such as NAD+ or NADP+ are used. The calibration curve is linear in the range of 0.1 to 4.0 g of ethanol per liter. Assays of 45 samples of serum having ethanol values ranging from 0.4 to 3.2 g/L by the described technique and a gas-chromatographic method gave respective means of 1.734 and 1.732 g/L (r = 0.954).

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Whyte, B. J., and P. A. Castelfranco. "Further observations on the Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase system." Biochemical Journal 290, no. 2 (March 1, 1993): 355–59. http://dx.doi.org/10.1042/bj2900355.

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The Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase was strongly inhibited by CN- and N3- in a reconstituted system, but was inhibited slightly or not at all by the same reagents in intact developing chloroplasts. Known inhibitors of cytochrome P-450 processes showed no consistent effect. Benzoquinone and quinol, which can give rise to the same semiquinone by one-electron redox events, were strong inhibitors of the cyclase. It was previously shown that O2 and a source of electrons are required in the cyclization process. The substrates for the dehydrogenases of the pentose phosphate pathway (glucose 6-phosphate and 6-phosphogluconate) were effective reductants in the reconstituted system with supernatant that had been dialysed or passed through Sephadex G-50, in the absence of added NADP+. However, inhibitor studies suggested that the electrons from these sugar phosphates reached the cyclase system via NADPH. Therefore we infer the presence of protein-bound NADP+ that can be reduced by glucose 6-phosphate and 6-phosphogluconate and donate reducing equivalents to the cyclase system. This bound NADPH pool may be particularly effective in the cyclization process, owing to channeling. These findings are discussed in relation to the results of a companion paper [Whyte and Castelfranco (1993) Biochem. J. 290, 361-367] on the breakdown of chloroplast pigments in the same reconstituted system.

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Campbell, Ashley C., Kyle M. Stiers, Julia S. Martin Del Campo, Ritcha Mehra-Chaudhary, Pablo Sobrado, and John J. Tanner. "Trapping conformational states of a flavin-dependent N-monooxygenase in crystallo reveals protein and flavin dynamics." Journal of Biological Chemistry 295, no. 38 (July 28, 2020): 13239–49. http://dx.doi.org/10.1074/jbc.ra120.014750.

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The siderophore biosynthetic enzyme A (SidA) ornithine hydroxylase from Aspergillus fumigatus is a fungal disease drug target involved in the production of hydroxamate-containing siderophores, which are used by the pathogen to sequester iron. SidA is an N-monooxygenase that catalyzes the NADPH-dependent hydroxylation of l-ornithine through a multistep oxidative mechanism, utilizing a C4a-hydroperoxyflavin intermediate. Here we present four new crystal structures of SidA in various redox and ligation states, including the first structure of oxidized SidA without NADP(H) or l-ornithine bound (resting state). The resting state structure reveals a new out active site conformation characterized by large rotations of the FAD isoalloxazine around the C1–′C2′ and N10–C1′ bonds, coupled to a 10-Å movement of the Tyr-loop. Additional structures show that either flavin reduction or the binding of NADP(H) is sufficient to drive the FAD to the in conformation. The structures also reveal protein conformational changes associated with the binding of NADP(H) and l-ornithine. Some of these residues were probed using site-directed mutagenesis. Docking was used to explore the active site of the out conformation. These calculations identified two potential ligand-binding sites. Altogether, our results provide new information about conformational dynamics in flavin-dependent monooxygenases. Understanding the different active site conformations that appear during the catalytic cycle may allow fine-tuning of inhibitor discovery efforts.

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Gay, D. A., D. Schmeltz, E. Prestbo, M. Olson, T. Sharac, and R. Tordon. "The Atmospheric Mercury Network: measurement and initial examination of an ongoing atmospheric mercury record across North America." Atmospheric Chemistry and Physics Discussions 13, no. 4 (April 19, 2013): 10521–46. http://dx.doi.org/10.5194/acpd-13-10521-2013.

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Abstract. The National Atmospheric Deposition Program (NADP) developed and operates a collaborative network of atmospheric mercury monitoring sites based in North America – the Atmospheric Mercury Network (AMNet). The justification for the network was growing interest and demand from many scientists and policy makers for a robust database of measurements to improve model development, assess policies and programs, and improve estimates of mercury dry deposition. Many different agencies and groups support the network, including federal, state, tribal, and international governments, academic institutions, and private companies. AMNet has added two high elevation sites outside of continental North America in Hawaii and Taiwan because of new partnerships forged within NADP. Network sites measure concentrations of atmospheric mercury fractions using automated, continuous mercury speciation systems. The procedures that NADP developed for field operations, data management, and quality assurance ensure that the network makes scientifically valid and consistent measurements. AMNet reports concentrations of hourly gaseous elemental mercury (GEM), two-hour gaseous oxidized mercury (GOM), and two-hour particulate-bound mercury less than 2.5 microns in size (PBM2.5). As of January 2012, over 450 000 valid observations are available from 30 stations. The AMNet also collects ancillary meteorological data and information on land-use and vegetation, when available. We present atmospheric mercury data comparisons by time (3 yr) at 22 unique site locations. Highlighted are contrasting values for site locations across the network: urban versus rural, coastal versus high-elevation and the range of maximum observations. The data presented should catalyze the formation of many scientific questions that may be answered through further in-depth analysis and modeling studies of the AMNet database. All data and methods are publically available through an online database on the NADP website (http://nadp.isws.illinois.edu/amn/). Future network directions are to foster new network partnerships and continue to collect, quality assure, and post data, including dry deposition estimates, for each fraction.

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Gay, D. A., D. Schmeltz, E. Prestbo, M. Olson, T. Sharac, and R. Tordon. "The Atmospheric Mercury Network: measurement and initial examination of an ongoing atmospheric mercury record across North America." Atmospheric Chemistry and Physics 13, no. 22 (November 22, 2013): 11339–49. http://dx.doi.org/10.5194/acp-13-11339-2013.

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Abstract. The National Atmospheric Deposition Program (NADP) developed and operates a collaborative network of atmospheric-mercury-monitoring sites based in North America – the Atmospheric Mercury Network (AMNet). The justification for the network was growing interest and demand from many scientists and policy makers for a robust database of measurements to improve model development, assess policies and programs, and improve estimates of mercury dry deposition. Many different agencies and groups support the network, including federal, state, tribal, and international governments, academic institutions, and private companies. AMNet has added two high-elevation sites outside of continental North America in Hawaii and Taiwan because of new partnerships forged within NADP. Network sites measure concentrations of atmospheric mercury fractions using automated, continuous mercury speciation systems. The procedures that NADP developed for field operations, data management, and quality assurance ensure that the network makes scientifically valid and consistent measurements. AMNet reports concentrations of hourly gaseous elemental mercury (GEM), two-hour gaseous oxidized mercury (GOM), and two-hour particulate-bound mercury less than 2.5 microns in size (PBM2.5). As of January 2012, over 450 000 valid observations are available from 30 stations. AMNet also collects ancillary meteorological data and information on land use and vegetation, when available. We present atmospheric mercury data comparisons by time (3 yr) at 21 individual sites and instruments. Highlighted are contrasting values for site locations across the network: urban versus rural, coastal versus high elevation and the range of maximum observations. The data presented should catalyze the formation of many scientific questions that may be answered through further in-depth analysis and modeling studies of the AMNet database. All data and methods are publically available through an online database on the NADP website (http://nadp.sws.uiuc.edu/amn/). Future network directions are to foster new network partnerships and continue to collect, quality assure, and post data, including dry deposition estimates, for each fraction.

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Niu, Guoqi, Qi Guo, Jia Wang, Shun Zhao, Yikun He, and Lin Liu. "Structural basis for plant lutein biosynthesis from α-carotene." Proceedings of the National Academy of Sciences 117, no. 25 (June 8, 2020): 14150–57. http://dx.doi.org/10.1073/pnas.2001806117.

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Two cytochrome P450 enzymes, CYP97A3 and CYP97C1, catalyze hydroxylations of the β- and ε-rings of α-carotene to produce lutein. Chirality is introduced at the C-3 atom of both rings, and the reactions are both pro-3R–stereospecific. We determined the crystal structures of CYP97A3 in substrate-free and complex forms with a nonnatural substrate and the structure of CYP97C1 in a detergent-bound form. The structures of CYP97A3 in different states show the substrate channel and the structure of CYP97C1 bound with octylthioglucoside confirms the binding site for the carotenoid substrate. Biochemical assays confirm that the ferredoxin-NADP+reductase (FNR)–ferredoxin pair is used as the redox partner. Details of the pro-3Rstereospecificity are revealed in the retinal-bound CYP97A3 structure. Further analysis indicates that the CYP97B clan bears similarity to the β-ring–specific CYP97A clan. Overall, our research describes the molecular basis for the last steps of lutein biosynthesis.

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Huang, Yu Chu, Neil B. Grodsky, Tae-Kang Kim, and Roberta F. Colman. "Ligands of the Mn2+Bound to Porcine Mitochondrial NADP-Dependent Isocitrate Dehydrogenase, as Assessed by Mutagenesis†." Biochemistry 43, no. 10 (March 2004): 2821–28. http://dx.doi.org/10.1021/bi030253f.

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JELESAROV, Ilian, Antonio R. PASCALIS, Willem H. KOPPENOL, Masakazu HIRASAWA, David B. KNAFF, and Hans Rudolf BOSSHARD. "Ferredoxin binding site on ferredoxin: NADP+ reductase. Differential chemical modification of free and ferredoxin-bound enzyme." European Journal of Biochemistry 216, no. 1 (August 1993): 57–66. http://dx.doi.org/10.1111/j.1432-1033.1993.tb18116.x.

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KAVANAGH, Kathryn L., Mario KLIMACEK, Bernd NIDETZKY, and David K. WILSON. "Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases." Biochemical Journal 373, no. 2 (July 15, 2003): 319–26. http://dx.doi.org/10.1042/bj20030286.

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Xylose reductase (XR; AKR2B5) is an unusual member of aldo-keto reductase superfamily, because it is one of the few able to efficiently utilize both NADPH and NADH as co-substrates in converting xylose into xylitol. In order to better understand the basis for this dual specificity, we have determined the crystal structure of XR from the yeast Candida tenuis in complex with NAD+ to 1.80 Å resolution (where 1 Å=0.1 nm) with a crystallographic R-factor of 18.3%. A comparison of the NAD+- and the previously determined NADP+-bound forms of XR reveals that XR has the ability to change the conformation of two loops. To accommodate both the presence and absence of the 2′-phosphate, the enzyme is able to adopt different conformations for several different side chains on these loops, including Asn276, which makes alternative hydrogen-bonding interactions with the adenosine ribose. Also critical is the presence of Glu227 on a short rigid helix, which makes hydrogen bonds to both the 2′- and 3′-hydroxy groups of the adenosine ribose. In addition to changes in hydrogen-bonding of the adenosine, the ribose unmistakably adopts a 3′-endo conformation rather than the 2′-endo conformation seen in the NADP+-bound form. These results underscore the importance of tight adenosine binding for efficient use of either NADH or NADPH as a co-substrate in aldo-keto reductases. The dual specificity found in XR is also an important consideration in designing a high-flux xylose metabolic pathway, which may be improved with an enzyme specific for NADH.

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38

Larson, Steven B., Jesse A. Jones, and Alexander McPherson. "The structure of an iron-containing alcohol dehydrogenase from a hyperthermophilic archaeon in two chemical states." Acta Crystallographica Section F Structural Biology Communications 75, no. 4 (March 13, 2019): 217–26. http://dx.doi.org/10.1107/s2053230x19001201.

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An iron-containing alcohol dehydrogenase (FeADH) from the hyperthermophilic archaeonThermococcus thioreducenswas crystallized in unit cells belonging to space groupsP21,P212121andP43212, and the crystal structures were solved at 2.4, 2.1 and 1.9 Å resolution, respectively, by molecular replacement using the FeADH fromThermotoga maritima(Schwarzenbacheret al., 2004) as a model. In the monoclinic and orthorhombic crystals the dehydrogenase (molecular mass 41.5 kDa) existed as a dimer containing a twofold noncrystallographic symmetry axis, which was crystallographic in the tetragonal crystals. In the monoclinic and orthorhombic asymmetric units one molecule contained iron and an NADP molecule, while the other did not. The tetragonal crystals lacked both iron and NADP. The structure is very similar to that of the FeADH fromT. maritima(average r.m.s. difference for Cαatoms of 1.8 Å for 341 aligned atoms). The iron, which is internally sequestered, is bound entirely by amino acids from one domain: three histidines and one aspartic acid. The coenzyme is in an extended conformation, a feature that is common to the large superfamily of NADH-dependent dehydrogenases that share a classical nucleotide-binding domain. A long broad tunnel passes entirely through the enzyme between the two domains, completely encapsulating the coenzyme.

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Okai, Masahiko, Norio Kudo, Woo Cheol Lee, Masayuki Kamo, Koji Nagata, and Masaru Tanokura. "Crystal Structures of the Short-Chain Flavin Reductase HpaC fromSulfolobus tokodaiiStrain 7 in Its Three States: NAD(P)+-Free, NAD+-Bound, and NADP+-Bound†,‡." Biochemistry 45, no. 16 (April 2006): 5103–10. http://dx.doi.org/10.1021/bi052313i.

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40

Ehrlich, Robert S., and Roberta F. Colman. "Ionization of isocitrate bound to pig heart NADP+-dependent isocitrate dehydrogenase: 13C NMR study of substrate binding." Biochemistry 26, no. 12 (June 16, 1987): 3461–66. http://dx.doi.org/10.1021/bi00386a032.

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Matthijs, Hans C. P., Deborah Moore, Sean J. Coughlan, and Geoffrey Hind. "Purification of membrane-bound ferredoxin: NADP+ oxidoreductase and of plastocyanin from a detergent extract of washed thylakoids." Photosynthesis Research 12, no. 3 (1987): 273–81. http://dx.doi.org/10.1007/bf00055127.

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42

Serra, Esteban C., Adriana R. Krapp, Jorgelina Ottado, Mario F. Feldman, Eduardo A. Ceccarelli, and Néstor Carrillo. "The Precursor of Pea Ferredoxin-NADP+Reductase Synthesized inEscherichia coliContains Bound FAD and Is Transported into Chloroplasts." Journal of Biological Chemistry 270, no. 34 (August 25, 1995): 19930–35. http://dx.doi.org/10.1074/jbc.270.34.19930.

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43

Wang, Zixi, Lanfen Li, Yu-Hui Dong, and Xiao-Dong Su. "Structural and biochemical characterization of MdaB from cariogenicStreptococcus mutansreveals an NADPH-specific quinone oxidoreductase." Acta Crystallographica Section D Biological Crystallography 70, no. 4 (March 19, 2014): 912–21. http://dx.doi.org/10.1107/s1399004713033749.

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Thesmu.1420 gene from the cariogenic pathogenStreptococcus mutansencodes a putative protein which has sequence homology to NQO [NAD(P)H:quinone oxidoreductase] family members, including mammalian NQO and bacterial MdaB (modulator of drug activity B). NQO can detoxify quinones by converting them to hydroquinones and prevent the generation of reactive oxygen species. Thus, comprehensive studies on Smu.1420 will be important for uncovering the antioxidation and antidrug mechanisms ofS. mutans. Here, the catalytic properties of Smu.1420 have been characterized, and its structure was determined in complexes with NADP+and menadione, respectively. Smu.1420 binds menadione directly and exhibits a pronounced preference for NADPH over NADH as a substrate, demonstrating that it is an NADPH-specific quinone oxidoreductase. The structure of Smu.1420 shows a compact homodimer with two substrate pockets located in the cleft of the dimer interface. The nicotinamide moiety of NADP+is bound on top of the isoalloxazine moiety of the FAD cofactor and overlaps with the binding site of menadione, suggesting a hydride-transfer process from NADPH to FAD and then to menadione. Two strongly basic patches near the substrate pocket are expected to confer the preference for NADPH over NADH. These studies shed light on future drug development against the cariogenic pathogenS. mutans.

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MEULLER, Johan, Junwei ZHANG, Cynthia HOU, Philip D. BRAGG, and Jan RYDSTRÖM. "Properties of a cysteine-free proton-pumping nicotinamide nucleotide transhydrogenase." Biochemical Journal 324, no. 2 (June 1, 1997): 681–87. http://dx.doi.org/10.1042/bj3240681.

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Nicotinamide nucleotide transhydrogenase from Escherichia coli was investigated with respect to the roles of its cysteine residues. This enzyme contains seven cysteines, of which five are located in the α subunit and two are in the β subunit. All cysteines were replaced by site-directed mutagenesis. The final construct (αC292T, αC339T, αC395S, αC397T, αC435S, βC147S, βC260S) was inserted normally in the membrane and underwent the normal NADPH-dependent conformational change of the β subunit to a trypsin-sensitive state. Reduction of NADP+ by NADH driven by ATP hydrolysis or respiration was between 32% and 65% of the corresponding wild-type activities. Likewise, the catalytic and proton pumping activities of the purified cysteine-free enzyme were at least 30% of the purified wild-type enzyme activities. The H+/H- ratio for both enzymes was 0.5, although the cysteine-free enzyme appeared to be more stable than the wild-type enzyme in proteoliposomes. No bound NADP(H) was detected in the enzymes. Modification of transhydrogenase by diethyl pyrocarbonate and the subsequent inhibition of the enzyme were unaffected by removal of the cysteines, indicating a lack of involvement of cysteines in this process. Replacement of cysteine residues in the α subunit resulted in no or little change in activity, suggesting that the basis for the decreased activity was probably the modification of the conserved β-subunit residue Cys-260 or (less likely) the non-conserved β-subunit residue Cys-147. It is concluded that the cysteine-free transhydrogenase is structurally and mechanistically very similar to the wild-type enzyme, with minor modifications of the properties of the NADP(H) site, possibly mediated by the βC260S mutation. The cysteine-free construct will be a valuable tool for studying structure–function relationships of transhydrogenases.

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Beslin, A., M. P. Vié, J. P. Blondeau, and J. Francon. "Identification by photoaffinity labelling of a pyridine nucleotide-dependent tri-iodothyronine-binding protein in the cytosol of cultured astroglial cells." Biochemical Journal 305, no. 3 (February 1, 1995): 729–37. http://dx.doi.org/10.1042/bj3050729.

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High-affinity 3,3′,5-tri-iodo-L-thyronine (T3) binding (Kd approximately 0.3 nM) to the cytosol of cultured rat astroglial cells was strongly activated in the presence of pyridine nucleotides. A 35 kDa pyridine nucleotide-dependent T3-binding polypeptide (35K-TBP) was photoaffinity labelled using underivatized [125I]T3 in the presence of pyridine nucleotides and the free-radical scavenger dithiothreitol. Maximum activations of T3 binding and 35K-TBP photolabelling were obtained at approx. 1 x 10(-7) M NADP+ or NADPH, or 1 x 10(-4) M NADH. NAD+ and other nucleotides were without effect. NADPH is the form which activates T3 binding and 35K-TBP photolabelling, since cytosol contains NADP(+)-reducing activity, and the activation of both processes in the presence of NADPH and NADP+ was prevented by an exogenous NADPH oxidation system. NADPH behaved as an allosteric activator of T3 binding. The NADPH oxidation system promoted the release of bound T3 in the absence of any change in the total concentration of the hormone. The 35K-TBP photolabelling and [125I]T3 binding were similarly inhibited by non-radioactive T3 (half-maximum effect at 0.5-1.0 nM T3). The concentrations of iodothyronine analogues that inhibited both processes were correlated (3,3′,5-tri-iodo-D-thyronine > or = T3 > L-thyroxine > tri-iodothyroacetic acid > 3,3′5′-tri-iodo-L-thyronine). Molecular sieving and density-gradient centrifugation of cytosol identified a 65 kDa T3-binding entity, which included the 35K-TBP. These results indicate that 35K-TBP is the cytosolic entity involved in the pyridine nucleotide-dependent T3 binding, and suggest that the sequestration and release of intracellular thyroid hormones are regulated by the redox state of astroglial cell compartment(s).

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Tait, G. H., C. J. Newton, M. J. Reed, and V. H. T. James. "Multiple forms of 17β-hydroxysteroid oxidoreductase in human breast tissue." Journal of Molecular Endocrinology 2, no. 1 (January 1989): 71–80. http://dx.doi.org/10.1677/jme.0.0020071.

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ABSTRACT 17β-Hydroxysteroid oxidoreductase, the enzyme that catalyses the interconversion of oestradiol and oestrone, is known to be present in human breast tissue. However, it is not known whether one or more forms of the enzyme is present. Homogenates of breast adipose tissue and breast glandular tissue were fractionated and fractions assayed in the oxidative direction with NAD+ and NADP+ as coenzymes, and in the reductive direction with NADH and NADPH as coenzymes. Ultracentrifugation of homogenates showed that there was membrane-bound activity and soluble activity. The soluble activity was due to a number of forms of the enzyme with different molecular weights, three in breast adipose tissue and two in breast glandular tissue, as shown by fractionation with (NH4)2SO4 followed by chromatography on Sephadex G-200. The forms of the enzyme isolated differed in their affinities for substrates and coenzymes and in the relative rates at which they catalysed the oxidative and reductive reactions. Preliminary experiments with breast tumours showed that they also contained membrane-bound activity and more than one soluble form of the enzyme.

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PETSCHACHER, Barbara, Stefan LEITGEB, Kathryn L. KAVANAGH, David K. WILSON, and Bernd NIDETZKY. "The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography." Biochemical Journal 385, no. 1 (December 14, 2004): 75–83. http://dx.doi.org/10.1042/bj20040363.

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CtXR (xylose reductase from the yeast Candida tenuis; AKR2B5) can utilize NADPH or NADH as co-substrate for the reduction of D-xylose into xylitol, NADPH being preferred approx. 33-fold. X-ray structures of CtXR bound to NADP+ and NAD+ have revealed two different protein conformations capable of accommodating the presence or absence of the coenzyme 2′-phosphate group. Here we have used site-directed mutagenesis to replace interactions specific to the enzyme–NADP+ complex with the aim of engineering the co-substrate-dependent conformational switch towards improved NADH selectivity. Purified single-site mutants K274R (Lys274→Arg), K274M, K274G, S275A, N276D, R280H and the double mutant K274R–N276D were characterized by steady-state kinetic analysis of enzymic D-xylose reductions with NADH and NADPH at 25 °C (pH 7.0). The results reveal between 2- and 193-fold increases in NADH versus NADPH selectivity in the mutants, compared with the wild-type, with only modest alterations of the original NADH-linked xylose specificity and catalytic-centre activity. Catalytic reaction profile analysis demonstrated that all mutations produced parallel effects of similar magnitude on ground-state binding of coenzyme and transition state stabilization. The crystal structure of the double mutant showing the best improvement of coenzyme selectivity versus wild-type and exhibiting a 5-fold preference for NADH over NADPH was determined in a binary complex with NAD+ at 2.2 Å resolution.

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Bennett, Melanie J., Ross H. Albert, Joseph M. Jez, Haiching Ma, Trevor M. Penning, and Mitchell Lewis. "Steroid recognition and regulation of hormone action: crystal structure of testosterone and NADP+ bound to 3α-hydroxysteroid/dihydrodiol dehydrogenase." Structure 5, no. 6 (June 1997): 799–812. http://dx.doi.org/10.1016/s0969-2126(97)00234-7.

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Serrano, Aurelio, Fernando C. Soncini, and Rubén H. Vallejos. "Localization and Quantitative Determination of Ferredoxin-NADP+ Oxidoreductase, a Thylakoid-Bound Enzyme in the Cyanobacterium Anabaena sp. Strain 7119." Plant Physiology 82, no. 2 (October 1, 1986): 499–502. http://dx.doi.org/10.1104/pp.82.2.499.

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Sugishima, Masakazu, Kei Wada, and Keiichi Fukuyama. "Recent Advances in the Understanding of the Reaction Chemistries of the Heme Catabolizing Enzymes HO and BVR Based on High Resolution Protein Structures." Current Medicinal Chemistry 27, no. 21 (June 15, 2020): 3499–518. http://dx.doi.org/10.2174/0929867326666181217142715.

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Abstract:

In mammals, catabolism of the heme group is indispensable for life. Heme is first cleaved by the enzyme Heme Oxygenase (HO) to the linear tetrapyrrole Biliverdin IXα (BV), and BV is then converted into bilirubin by Biliverdin Reductase (BVR). HO utilizes three Oxygen molecules (O2) and seven electrons supplied by NADPH-cytochrome P450 oxidoreductase (CPR) to open the heme ring and BVR reduces BV through the use of NAD(P)H. Structural studies of HOs, including substrate-bound, reaction intermediate-bound, and several specific inhibitor-bound forms, reveal details explaining substrate binding to HO and mechanisms underlying-specific HO reaction progression. Cryo-trapped structures and a time-resolved spectroscopic study examining photolysis of the bond between the distal ligand and heme iron demonstrate how CO, produced during the HO reaction, dissociates from the reaction site with a corresponding conformational change in HO. The complex structure containing HO and CPR provides details of how electrons are transferred to the heme-HO complex. Although the tertiary structure of BVR and its complex with NAD+ was determined more than 10 years ago, the catalytic residues and the reaction mechanism of BVR remain unknown. A recent crystallographic study examining cyanobacterial BVR in complex with NADP+ and substrate BV provided some clarification regarding these issues. Two BV molecules are bound to BVR in a stacked manner, and one BV may assist in the reductive catalysis of the other BV. In this review, recent advances illustrated by biochemical, spectroscopic, and crystallographic studies detailing the chemistry underlying the molecular mechanism of HO and BVR reactions are presented.

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Journal articles on the topic 'NADP-bound'

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