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1
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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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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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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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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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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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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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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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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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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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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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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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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Steuber, Julia, Walter Krebs, Michael Bott, and Peter Dimroth. "A Membrane-Bound NAD(P)+-Reducing Hydrogenase Provides Reduced Pyridine Nucleotides during Citrate Fermentation by Klebsiella pneumoniae." Journal of Bacteriology 181, no. 1 (January 1, 1999): 241–45. http://dx.doi.org/10.1128/jb.181.1.241-245.1999.
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ABSTRACT During anaerobic growth of Klebsiella pneumoniae on citrate, 9.4 mmol of H2/mol of citrate (4-kPa partial pressure) was formed at the end of growth besides acetate, formate, and CO2. Upon addition of NiCl2 (36 μM) to the growth medium, hydrogen formation increased about 36% to 14.8 mmol/mol of citrate (6 kPa), and the cell yield increased about 15%. Cells that had been harvested and washed under anoxic conditions exhibited an H2-dependent formation of NAD(P)H in vivo. The reduction of internal NAD(P)+ was also achieved by the addition of formate. In crude extracts, the H2:NAD+oxidoreductase activity was 0.13 μmol min−1mg−1, and 76% of this activity was found in the washed membrane fraction. The highest specific activities of the membrane fraction were observed in 50 mM potassium phosphate, with 1.6 μmol of NADPH formed min−1 mg−1 at pH 7.0 and 1.7 μmol of NADH formed min−1 mg−1 at pH 9.5. In the presence of the protonophore carbonyl cyanidem-chlorophenylhydrazone and the Na+/H+ antiporter monensin, the H2-dependent reduction of NAD+ by membrane vesicles decreased only slightly (about 16%). The NADP+- or NAD+-reducing hydrogenases were solubilized from the membranes with the detergent lauryldimethylamine-N-oxide or Triton X-100. NAD(P)H formation with H2 as electron donor, therefore, does not depend on an energized state of the membrane. It is proposed that hydrogen which is formed by K. pneumoniaeduring citrate fermentation is recaptured by a novel membrane-bound, oxygen-sensitive H2:NAD(P)+ oxidoreductase that provides reducing equivalents for the synthesis of cell material.
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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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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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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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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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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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Kratzer, Regina, and Bernd Nidetzky. "Electrostatic stabilization in a pre-organized polar active site: the catalytic role of Lys-80 in Candida tenuis xylose reductase (AKR2B5) probed by site-directed mutagenesis and functional complementation studies." Biochemical Journal 389, no. 2 (July 5, 2005): 507–15. http://dx.doi.org/10.1042/bj20050167.
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Lys-80 of Candida tenuis xylose reductase (AKR2B5) is conserved throughout the aldo–keto reductase protein superfamily and may prime the nearby Tyr-51 for general acid catalysis to NAD(P)H-dependent carbonyl group reduction. We have examined the catalytic significance of side-chain substitutions in two AKR2B5 mutants, Lys-80→Ala (K80A) and Asp-46→Asn Lys-80→Ala (D46N K80A), using steady-state kinetic analysis and restoration of activity with external amines. Binding of NAD+ (Kd=24 μM) and NADP+ (Kd=0.03 μM) was 10- and 40-fold tighter in K80A than the wild-type enzyme, whereas binding of NADH (Kd=51 μM) and NADPH (Kd=19 μM) was weakened 2- and 16-fold in this mutant respectively. D46N K80A bound NAD(P)H and NAD(P)+ uniformly approx. 5-fold less tightly than the wild-type enzyme. The second-order rate constant for non-covalent restoration of NADH-dependent reductase activity (kmax/Kamine) by protonated ethylamine was 0.11 M−1·s−1 for K80A, whereas no detectable rescue occurred for D46N K80A. After correction for effects of side-chain hydrophobicity, we obtained a linear free energy relationship of log (kmax/Kamine) and amine group pKa (slope=+0.29; r2=0.93) at pH 7.0. pH profiles of log (kcat/Km) for carbonyl group reduction by wild-type and D46N K80A revealed identical and kinetically unperturbed pKa values of 8.50 (±0.20). Therefore the protonated side chain of Lys-80 is not an essential activator of general acid catalysis by AKR2B5. Stabilized structurally through the salt-link interaction with the negatively charged Asp-46, it is proposed to pull the side chain of Tyr-51 into the catalytic position, leading to a preorganized polar environment of overall neutral charge, in which approximation of uncharged reactive groups is favoured and thus hydride transfer from NAD(P)H is strongly preferred. Lys-80 affects further the directional preference of AKR2B5 for NAD(P)H-dependent reduction by increasing NAD(P)H compared with NAD(P)+-binding selectivity.
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Bronstein, M. E., and N. M. Chikhladze. "Histochemical characteristics of aldosteromas and adrenal tissue of patients with primary hyperaldosteronism." Problems of Endocrinology 40, no. 1 (February 15, 1994): 26–30. http://dx.doi.org/10.14341/probl11291.
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The authors analyze the data of histochemical examinations of 30 patients with tumorous and nontu- morous forms of low-renin hyperaldosteronism, as well as the findings of clinical and hormonal examinations of these patients. The activities of enzymes involved in steroidogenesis (3-pOSD, NAD and NADP tetrazoleum reductases, G-6-PD) were measured, as were levels and distribution patterns of lipids involved in steroidogenesis (free and bound cholesterol, etc.). A high functional activity of aldosteromas of any structure was proved, and a trend to increase of steroidogenic activity of malignant aldosteromas demonstrated. Atrophic changes in the cortex adjacent to aldosteroma were found to involve no reduction of adrenal tissue functional activity. In contrast to this, hyperplastic changes in the cortex adjacent to adenoma were characterized by a lower level of functional activity as against adrenal tissue activity in nontumorous hyperaldosteronism. The authors come to a conclusion that adrenocortical elements hyperplasia associated with some aldosteromas does not participate in aldosterone hyperproduction whereas hyperaldosteronism in nontumorous variants is caused by cortical cell hyperplasia and hormone hyperproduction.
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Chalmers, R. M., and C. A. Fewson. "Purification and characterization of benzaldehyde dehydrogenase I from Acinetobacter calcoaceticus." Biochemical Journal 263, no. 3 (November 1, 1989): 913–19. http://dx.doi.org/10.1042/bj2630913.
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Benzaldehyde dehydrogenase I was purified from Acinetobacter calcoaceticus by DEAE-Sephacel, phenyl-Sepharose and f.p.l.c. gel-filtration chromatography. The enzyme was homogeneous and completely free from the isofunctional enzyme benzaldehyde dehydrogenase II, as judged by denaturing and non-denaturing polyacrylamide-gel electrophoresis. The subunit Mr value was 56,000 (determined by SDS/polyacrylamide-gel electrophoresis). Estimations of the native Mr value by gel-filtration chromatography gave values of 141,000 with a f.p.l.c. Superose 6 column, but 219,000 with Sephacryl S300. Chemical cross-linking of the enzyme subunits indicated that the enzyme is tetrameric. Benzaldehyde dehydrogenase I was activated more than 100-fold by K+, Rb+ and NH4+, and the apparent Km for K+ was 11.2 mM. The pH optimum in the presence of K+ was 9.5 and the pI of the enzyme was 5.55. The apparent Km values for benzaldehyde and NAD+ were 0.69 microM and 96 microM respectively, and the maximum velocity was approx. 110 mumol/min per mg of protein. Various substituted benzaldehydes were oxidized at significant rates, and NADP+ was also used as cofactor, although much less effectively than NAD+. Benzaldehyde dehydrogenase I had an NAD+-activated esterase activity with 4-nitrophenol acetate as substrate, and the dehydrogenase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group. Some of the properties of the enzyme are compared with those of other aldehyde dehydrogenases, specifically the very similar isofunctional enzyme benzaldehyde dehydrogenase II from the same organism.
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Di Luccio, Eric, Robert A. Elling, and David K. Wilson. "Identification of a novel NADH-specific aldo-keto reductase using sequence and structural homologies." Biochemical Journal 400, no. 1 (October 27, 2006): 105–14. http://dx.doi.org/10.1042/bj20060660.
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The AKRs (aldo-keto reductases) are a superfamily of enzymes which mainly rely on NADPH to reversibly reduce various carbonyl-containing compounds to the corresponding alcohols. A small number have been found with dual NADPH/NADH specificity, usually preferring NADPH, but none are exclusive for NADH. Crystal structures of the dual-specificity enzyme xylose reductase (AKR2B5) indicate that NAD+ is bound via a key interaction with a glutamate that is able to change conformations to accommodate the 2′-phosphate of NADP+. Sequence comparisons suggest that analogous glutamate or aspartate residues may function in other AKRs to allow NADH utilization. Based on this, nine putative enzymes with potential NADH specificity were identified and seven genes were successfully expressed and purified from Drosophila melanogaster, Escherichia coli, Schizosaccharomyces pombe, Sulfolobus solfataricus, Sinorhizobium meliloti and Thermotoga maritima. Each was assayed for co-substrate dependence with conventional AKR substrates. Three were exclusive for NADPH (AKR2E3, AKR3F2 and AKR3F3), two were dual-specific (AKR3C2 and AKR3F1) and one was specific for NADH (AKR11B2), the first such activity in an AKR. Fluorescence measurements of the seventh protein indicated that it bound both NADPH and NADH but had no activity. Mutation of the aspartate into an alanine residue or a more mobile glutamate in the NADH-specific E. coli protein converted it into an enzyme with dual specificity. These results show that the presence of this carboxylate is an indication of NADH dependence. This should allow improved prediction of co-substrate specificity and provide a basis for engineering enzymes with altered co-substrate utilization for this class of enzymes.
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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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Daußmann, Thomas, Alexander Aivasidis, and Christian Wandrey. "Kinetic data and new enzymatic activities of Methanosarcina barkeri grown on methanol as the sole carbon source." Water Science and Technology 36, no. 6-7 (September 1, 1997): 175–82. http://dx.doi.org/10.2166/wst.1997.0589.
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The strictly anaerobic methanogen Methanosarcina barkeri strain Fusaro (DSM 804) was fermented on a 3-liter scale in a CSTR (continuous stirred tank reactor) to obtain almost 10 g biomass dry weight per day. The generation time of the microorganism with methanol as the sole carbon source was 6.0 h, the maximal growth rate 0.115 h−1 correspondingly. In cell-free extracts from the fermented organism active hydrogenases were found. They reduced methyl viologen (MV⊕⊕), an artificial electron acceptor, molecular hydrogen being the substrate. This reaction was used to get electrochemical energy in a biochemical fuel cell. The maximum output of this fuel cell was 2.6 mW. Furthermore, cell-free extracts from M. barkeri showed alcohol dehydrogenase activity under aerobic conditions when N,N′-dimethyl-4-nitrosoaniline (NDMA) was used as an artificial electron acceptor. The NDMA-dependent alcohol dehydrogenase (NDMA-ADH) was purified to homogeneity by column chromatography. It is a homodimeric enzyme consisting of subunits of 45 kDa, the native molecular mass is about 87 kDa. The enzyme is independent of free coenzyme such as NAD, NADP, FMN, FAD and F420, but it possesses a tightly but noncovalently bound NADP(H) cofactor. The purified enzyme exhibited activity only with primary alcohols including aromatic alcohols, but methanol was not accepted. It also catalyzed the stoichiometric dismutation of aldehydes, especially long-chain aldehydes, to form a half mol of each of the corresponding alcohol and acid without addition of an electron carrier. NDMA-ADH from M. barkeri is a novel type of nicotinoprotein in methanogenic bacteria.
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Stolarska, Ewelina, Karolina Bilska, Natalia Wojciechowska, Agnieszka Bagniewska-Zadworna, Pascal Rey, and Ewa M. Kalemba. "Integration of MsrB1 and MsrB2 in the Redox Network during the Development of Orthodox and Recalcitrant Acer Seeds." Antioxidants 9, no. 12 (December 9, 2020): 1250. http://dx.doi.org/10.3390/antiox9121250.
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Two related tree species, Norway maple (Acer platanoides L.) and sycamore (Acer pseudoplatanus L.), produce desiccation-tolerant (orthodox) and desiccation-sensitive (recalcitrant) seeds, respectively. We compared the seeds of these two species to characterize the developmentally driven changes in the levels of peptide-bound methionine sulfoxide (MetO) and the abundance of methionine sulfoxide reductases (Msrs) B1 and B2, with respect to the cellular redox environment. Protein oxidation at the Met level was dynamic only in Norway maple seeds, and the reduced MsrB2 form was detected only in this species. Cell redox status, characterized by the levels of reduced and oxidized ascorbate, glutathione, and nicotinamide adenine dinucleotide (NAD)/phosphate (NADP), was clearly more reduced in the Norway maple seeds than in the sycamore seeds. Clear correlations between MetO levels, changes in water content and redox status were reported in orthodox Acer seeds. The abundance of Msrs was correlated in both species with redox determinants, mainly ascorbate and glutathione. Our data suggest that MsrB2 is associated with the acquisition of desiccation tolerance and that ascorbate might be involved in the redox pathway enabling the regeneration of Msr via intermediates that are not known yet.
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Benson, David R., Scott Lovell, Nurjahan Mehzabeen, Nadezhda Galeva, Anne Cooper, Philip Gao, Kevin P. Battaile, and Hao Zhu. "Crystal structures of the naturally fused CS and cytochrome b 5 reductase (b 5R) domains of Ncb5or reveal an expanded CS fold, extensive CS–b 5R interactions and productive binding of the NAD(P)+ nicotinamide ring." Acta Crystallographica Section D Structural Biology 75, no. 7 (June 26, 2019): 628–38. http://dx.doi.org/10.1107/s205979831900754x.
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Ncb5or (NADH-cytochrome b 5 oxidoreductase), a cytosolic ferric reductase implicated in diabetes and neurological diseases, comprises three distinct domains, cytochrome b 5 (b 5) and cytochrome b 5 reductase (b 5R) domains separated by a CHORD–Sgt1 (CS) domain, and a novel 50-residue N-terminal region. Understanding how interdomain interactions in Ncb5or facilitate the shuttling of electrons from NAD(P)H to heme, and how the process compares with the microsomal b 5 (Cyb5A) and b 5R (Cyb5R3) system, is of interest. A high-resolution structure of the b 5 domain (PDB entry 3lf5) has previously been reported, which exhibits substantial differences in comparison to Cyb5A. The structural characterization of a construct comprising the naturally fused CS and b 5R domains with bound FAD and NAD+ (PDB entry 6mv1) or NADP+ (PDB entry 6mv2) is now reported. The structures reveal that the linker between the CS and b 5R cores is more ordered than predicted, with much of it extending the β-sandwich motif of the CS domain. This limits the flexibility between the two domains, which recognize one another via a short β-sheet motif and a network of conserved side-chain hydrogen bonds, salt bridges and cation–π interactions. Notable differences in FAD–protein interactions in Ncb5or and Cyb5R3 provide insight into the selectivity for docking of their respective b 5 redox partners. The structures also afford a structural explanation for the unusual ability of Ncb5or to utilize both NADH and NADPH, and represent the first examples of native, fully oxidized b 5R family members in which the nicotinamide ring of NAD(P)+ resides in the active site. Finally, the structures, together with sequence alignments, show that the b 5R domain is more closely related to single-domain Cyb5R proteins from plants, fungi and some protists than to Cyb5R3 from animals.
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Wang, Feng, Paras Jain, Gulcin Gulten, Zhen Liu, Yicheng Feng, Krishna Ganesula, Alifiya S. Motiwala, et al. "Mycobacterium tuberculosis Dihydrofolate Reductase Is Not a Target Relevant to the Antitubercular Activity of Isoniazid." Antimicrobial Agents and Chemotherapy 54, no. 9 (June 21, 2010): 3776–82. http://dx.doi.org/10.1128/aac.00453-10.
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ABSTRACT Mycobacterium tuberculosis enoyl-acyl-ACP reductase (InhA) has been demonstrated to be the primary target of isoniazid (INH). Recently, it was postulated that M. tuberculosis dihydrofolate reductase (DHFR) is also a target of INH, based on the findings that a 4R-INH-NADP adduct synthesized from INH by a nonenzymatic approach showed strong inhibition of DHFR in vitro, and overexpression of M. tuberculosis dfrA in M. smegmatis conferred a 2-fold increase of resistance to INH. In the present study, a plasmid expressing M. tuberculosis dfrA was transformed into M. smegmatis and M. tuberculosis strains, respectively. The transformant strains were tested for their resistance to INH. Compared to the wild-type strains, overexpression of dfrA in M. smegmatis and M. tuberculosis did not confer any resistance to INH based on the MIC values. Similar negative results were obtained with 14 other overexpressed proteins that have been proposed to bind some form of INH-NAD(P) adduct. An Escherichia coli cell-based system was designed that allowed coexpression of both M. tuberculosis katG and dfrA genes in the presence of INH. The DHFR protein isolated from the experimental sample was not found bound with any INH-NADP adduct by enzyme inhibition assay and mass spectroscopic analysis. We also used whole-genome sequencing to determine whether polymorphisms in dfrA could be detected in six INH-resistant clinical isolates known to lack mutations in inhA and katG, but no such mutations were found. The dfrA overexpression experiments, together with the biochemical and sequencing studies, conclusively demonstrate that DHFR is not a target relevant to the antitubercular activity of INH.
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BURDETTE, Douglas S., Francesco SECUNDO, Robert S. PHILLIPS, Jun DONG, Robert A. SCOTT, and J. Gregory ZEIKUS. "Biophysical and mutagenic analysis of Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase activity and specificity." Biochemical Journal 326, no. 3 (September 15, 1997): 717–24. http://dx.doi.org/10.1042/bj3260717.
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The Thermoanaerobacter ethanolicus 39E adhB gene encoding the secondary-alcohol dehydrogenase (2° ADH) was overexpressed in Escherichia coli at more than 10% of total protein. The recombinant enzyme was purified in high yield (67%) by heat-treatment at 85 °C and (NH4)2SO4 precipitation. Site-directed mutants (C37S, H59N, D150N, D150E and D150C were analysed to test the peptide sequence comparison-based predictions of amino acids responsible for putative catalytic Zn binding. X-ray absorption spectroscopy confirmed the presence of a protein-bound Zn atom with ZnS1(imid)1(N,O)3 co-ordination sphere. Inductively coupled plasma atomic emission spectrometry measured 0.48 Zn atoms per wild-type 2° ADH subunit. The C37S, H59N and D150N mutant enzymes bound only 0.11, 0.13 and 0.33 Zn per subunit respectively, suggesting that these residues are involved in Zn liganding. The D150E and D150C mutants retained 0.47 and 1.2 Zn atoms per subunit, indicating that an anionic side-chain moiety at this position preserves the bound Zn. All five mutant enzymes had ⩽ 3% of wild-type catalytic activity, suggesting that the T. ethanolicus 2° ADH requires a properly co-ordinated catalytic Zn atom. The His-59 and Asp-150 mutations also altered 2° ADH affinity for propan-2-ol over a 140-fold range, whereas the overall change in affinity for ethanol spanned a range of only 7-fold, supporting the importance of the metal in 2° ADH substrate binding. The lack of significant changes in cofactor affinity as a result of these catalytic Zn ligand mutations suggested that 2° ADH substrate-and cofactor-binding sites are structurally distinct. Altering Gly198 to Asp reduced the enzyme specific activity 2.7-fold, increased the Km(app) for NADP+ 225-fold, and decreased the Km(app) for NAD+ 3-fold, supporting the prediction that the enzyme binds nicotinamide cofactor in a Rossmann fold. Our data indicate therefore that, unlike the liver 1° ADH, the Rossmann-fold-containing T. ethanolicus 2° ADH binds its catalytic Zn atom using a sorbitol dehydrogenase-like Cys-His-Asp motif and does not bind a structural Zn atom.
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McFarlane, Jeffrey S., Cara L. Davis, and Audrey L. Lamb. "Staphylopine, pseudopaline, and yersinopine dehydrogenases: A structural and kinetic analysis of a new functional class of opine dehydrogenase." Journal of Biological Chemistry 293, no. 21 (April 4, 2018): 8009–19. http://dx.doi.org/10.1074/jbc.ra118.002007.
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Opine dehydrogenases (ODHs) from the bacterial pathogens Staphylococcus aureus, Pseudomonas aeruginosa, and Yersinia pestis perform the final enzymatic step in the biosynthesis of a new class of opine metallophores, which includes staphylopine, pseudopaline, and yersinopine, respectively. Growing evidence indicates an important role for this pathway in metal acquisition and virulence, including in lung and burn-wound infections (P. aeruginosa) and in blood and heart infections (S. aureus). Here, we present kinetic and structural characterizations of these three opine dehydrogenases. A steady-state kinetic analysis revealed that the three enzymes differ in α-keto acid and NAD(P)H substrate specificity and nicotianamine-like substrate stereoselectivity. The structural basis for these differences was determined from five ODH X-ray crystal structures, ranging in resolution from 1.9 to 2.5 Å, with or without NADP+ bound. Variation in hydrogen bonding with NADPH suggested an explanation for the differential recognition of this substrate by these three enzymes. Our analysis further revealed candidate residues in the active sites required for binding of the α-keto acid and nicotianamine-like substrates and for catalysis. This work reports the first structural kinetic analyses of enzymes involved in opine metallophore biosynthesis in three important bacterial pathogens of humans.
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Schut, Gerrit J., Stephanie L. Bridger, and Michael W. W. Adams. "Insights into the Metabolism of Elemental Sulfur by the Hyperthermophilic Archaeon Pyrococcus furiosus: Characterization of a Coenzyme A- Dependent NAD(P)H Sulfur Oxidoreductase." Journal of Bacteriology 189, no. 12 (April 20, 2007): 4431–41. http://dx.doi.org/10.1128/jb.00031-07.
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ABSTRACT The hyperthermophilic archaeon Pyrococcus furiosus uses carbohydrates as a carbon source and produces acetate, CO2, and H2 as end products. When S0 is added to a growing culture, within 10 min the rate of H2 production rapidly decreases and H2S is detected. After 1 hour cells contain high NADPH- and coenzyme A-dependent S0 reduction activity (0.7 units/mg, 85°C) located in the cytoplasm. The enzyme responsible for this activity was purified to electrophoretic homogeneity (specific activity, 100 units/mg) and is termed NAD(P)H elemental sulfur oxidoreductase (NSR). NSR is a homodimeric flavoprotein (M r, 100,000) and is encoded by PF1186. This designation was previously assigned to the gene encoding an enzyme that reduces coenzyme A disulfide, which is a side reaction of NSR. Whole-genome DNA microarray and quantitative PCR analyses showed that the expression of NSR is up-regulated up to sevenfold within 10 min of S0 addition. This primary response to S0 also involves the up-regulation (>16-fold) of a 13-gene cluster encoding a membrane-bound oxidoreductase (MBX). The cluster encoding MBX is proposed to replace the homologous 14-gene cluster that encodes the ferredoxin-oxidizing, H2-evolving membrane-bound hydrogenase (MBH), which is down-regulated >12-fold within 10 min of S0 addition. Although an activity for MBX could not be demonstrated, it is proposed to conserve energy by oxidizing ferredoxin and reducing NADP, which is used by NSR to reduce S0. A secondary response to S0 is observed 30 min after S0 addition and includes the up-regulation of genes encoding proteins involved in amino acid biosynthesis and iron metabolism, as well as two so-called sulfur-induced proteins termed SipA and SipB. This novel S0-reducing system involving NSR and MBX has been found so far only in the heterotrophic Thermococcales and is in contrast to the cytochrome- and quinone-based S0-reducing system in autotrophic archaea and bacteria.
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Leitgeb, Stefan, Barbara Petschacher, David K. Wilson, and Bernd Nidetzky. "Fine tuning of coenzyme specificity in family 2 aldo-keto reductases revealed by crystal structures of the Lys-274 → Arg mutant ofCandida tenuisxylose reductase (AKR2B5) bound to NAD+and NADP+." FEBS Letters 579, no. 3 (January 11, 2005): 763–67. http://dx.doi.org/10.1016/j.febslet.2004.12.063.
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Giddings, Lesley-Ann, George T. Lountos, Kang Woo Kim, Matthew Brockley, Danielle Needle, Scott Cherry, Joseph E. Tropea, and David S. Waugh. "Characterization of a broadly specific cadaverine N-hydroxylase involved in desferrioxamine B biosynthesis in Streptomyces sviceus." PLOS ONE 16, no. 3 (March 30, 2021): e0248385. http://dx.doi.org/10.1371/journal.pone.0248385.
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N-hydroxylating flavin-dependent monooxygenases (FMOs) are involved in the biosynthesis of hydroxamate siderophores, playing a key role in microbial virulence. Herein, we report the first structural and kinetic characterization of a novel alkyl diamine N-hydroxylase DesB from Streptomyces sviceus (SsDesB). This enzyme catalyzes the first committed step in the biosynthesis of desferrioxamine B, a clinical drug used to treat iron overload disorders. X-ray crystal structures of the SsDesB holoenzyme with FAD and the ternary complex with bound NADP+ were solved at 2.86 Å and 2.37 Å resolution, respectively, providing a structural view of the active site environment. SsDesB crystallized as a tetramer and the structure of the individual protomers closely resembles the structures of homologous N-hydroxylating FMOs from Erwinia amylovora (DfoA), Pseudomonas aeruginosa (PvdA), and Aspergillus fumigatus (SidA). Using NADPH oxidation, oxygen consumption, and product formation assays, kinetic parameters were determined for various substrates with SsDesB. SsDesB exhibited typical saturation kinetics with substrate inhibition at high concentrations of NAD(P)H as well as cadaverine. The apparent kcat values for NADPH in steady-state NADPH oxidation and oxygen consumption assays were 0.28 ± 0.01 s-1 and 0.24 ± 0.01 s-1, respectively. However, in product formation assays used to measure the rate of N-hydroxylation, the apparent kcat for NADPH (0.034 ± 0.008 s-1) was almost 10-fold lower under saturating FAD and cadaverine concentrations, reflecting an uncoupled reaction, and the apparent NADPH KM was 33 ± 24 μM. Under saturating FAD and NADPH concentrations, the apparent kcat and KM for cadaverine in Csaky assays were 0.048 ± 0.004 s-1 and 19 ± 9 μM, respectively. SsDesB also N-hydroxylated putrescine, spermidine, and L-lysine substrates but not alkyl (di)amines that were branched or had fewer than four methylene units in an alkyl chain. These data demonstrate that SsDesB has wider substrate scope compared to other well-studied ornithine and lysine N-hydroxylases, making it an amenable biocatalyst for the production of desferrioxamine B, derivatives, and other N-substituted products.
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Lhor, Mustapha, and Christian Salesse. "Retinol dehydrogenases: Membrane-bound enzymes for the visual function." Biochemistry and Cell Biology 92, no. 6 (December 2014): 510–23. http://dx.doi.org/10.1139/bcb-2014-0082.
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Retinoid metabolism is important for many physiological functions, such as differenciation, growth, and vision. In the visual context, after the absorption of light in rod photoreceptors by the visual pigment rhodopsin, 11-cis retinal is isomerized to all-trans retinal. This retinoid subsequently undergoes a series of modifications during the visual cycle through a cascade of reactions occurring in photoreceptors and in the retinal pigment epithelium. Retinol dehydrogenases (RDHs) are enzymes responsible for crucial steps of this visual cycle. They belong to a large family of proteins designated as short-chain dehydrogenases/reductases. The structure of these RDHs has been predicted using modern bioinformatics tools, which allowed to propose models with similar structures including a common Rossman fold. These enzymes undergo oxidoreduction reactions, whose direction is dictated by the preference and concentration of their individual cofactor (NAD(H)/NADP(H)). This review presents the current state of knowledge on functional and structural features of RDHs involved in the visual cycle as well as knockout models. RDHs are described as integral or peripheral enzymes. A topology model of the membrane binding of these RDHs via their N- and (or) C-terminal domain has been proposed on the basis of their individual properties. Membrane binding is a crucial issue for these enzymes because of the high hydrophobicity of their retinoid substrates.
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Alphonse, Sébastien, Ankan Banerjee, Swathi Dantuluri, Stewart Shuman, and Ranajeet Ghose. "NMR solution structures of Runella slithyformis RNA 2′-phosphotransferase Tpt1 provide insights into NAD+ binding and specificity." Nucleic Acids Research, April 21, 2021. http://dx.doi.org/10.1093/nar/gkab241.
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Abstract Tpt1, an essential component of the fungal and plant tRNA splicing machinery, catalyzes transfer of an internal RNA 2′-PO4 to NAD+ yielding RNA 2′-OH and ADP-ribose-1′,2′-cyclic phosphate products. Here, we report NMR structures of the Tpt1 ortholog from the bacterium Runella slithyformis (RslTpt1), as apoenzyme and bound to NAD+. RslTpt1 consists of N- and C-terminal lobes with substantial inter-lobe dynamics in the free and NAD+-bound states. ITC measurements of RslTpt1 binding to NAD+ (KD ∼31 μM), ADP-ribose (∼96 μM) and ADP (∼123 μM) indicate that substrate affinity is determined primarily by the ADP moiety; no binding of NMN or nicotinamide is observed by ITC. NAD+-induced chemical shift perturbations (CSPs) localize exclusively to the RslTpt1 C-lobe. NADP+, which contains an adenylate 2′-PO4 (mimicking the substrate RNA 2′-PO4), binds with lower affinity (KD ∼1 mM) and elicits only N-lobe CSPs. The RslTpt1·NAD+ binary complex reveals C-lobe contacts to adenosine ribose hydroxyls (His99, Thr101), the adenine nucleobase (Asn105, Asp112, Gly113, Met117) and the nicotinamide riboside (Ser125, Gln126, Asn163, Val165), several of which are essential for RslTpt1 activity in vivo. Proximity of the NAD+ β-phosphate to ribose-C1″ suggests that it may stabilize an oxocarbenium transition-state during the first step of the Tpt1-catalyzed reaction.
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Chornyi, Serhii, Lodewijk IJlst, Carlo W. T. van Roermund, Ronald J. A. Wanders, and Hans R. Waterham. "Peroxisomal Metabolite and Cofactor Transport in Humans." Frontiers in Cell and Developmental Biology 8 (January 11, 2021). http://dx.doi.org/10.3389/fcell.2020.613892.
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Peroxisomes are membrane-bound organelles involved in many metabolic pathways and essential for human health. They harbor a large number of enzymes involved in the different pathways, thus requiring transport of substrates, products and cofactors involved across the peroxisomal membrane. Although much progress has been made in understanding the permeability properties of peroxisomes, there are still important gaps in our knowledge about the peroxisomal transport of metabolites and cofactors. In this review, we discuss the different modes of transport of metabolites and essential cofactors, including CoA, NAD+, NADP+, FAD, FMN, ATP, heme, pyridoxal phosphate, and thiamine pyrophosphate across the peroxisomal membrane. This transport can be mediated by non-selective pore-forming proteins, selective transport proteins, membrane contact sites between organelles, and co-import of cofactors with proteins. We also discuss modes of transport mediated by shuttle systems described for NAD+/NADH and NADP+/NADPH. We mainly focus on current knowledge on human peroxisomal metabolite and cofactor transport, but also include knowledge from studies in plants, yeast, fruit fly, zebrafish, and mice, which has been exemplary in understanding peroxisomal transport mechanisms in general.
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Abdel-Hady, Gamal Nasser, Takeshi Ikeda, Takenori Ishida, Hisakage Funabashi, Akio Kuroda, and Ryuichi Hirota. "Engineering Cofactor Specificity of a Thermostable Phosphite Dehydrogenase for a Highly Efficient and Robust NADPH Regeneration System." Frontiers in Bioengineering and Biotechnology 9 (April 1, 2021). http://dx.doi.org/10.3389/fbioe.2021.647176.
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Nicotinamide adenine dinucleotide phosphate (NADP)-dependent dehydrogenases catalyze a range of chemical reactions useful for practical applications. However, their dependence on the costly cofactor, NAD(P)H remains a challenge which must be addressed. Here, we engineered a thermotolerant phosphite dehydrogenase from Ralstonia sp. 4506 (RsPtxD) by relaxing the cofactor specificity for a highly efficient and robust NADPH regeneration system. The five amino acid residues, Cys174–Pro178, located at the C-terminus of β7-strand region in the Rossmann-fold domain of RsPtxD, were changed by site-directed mutagenesis, resulting in four mutants with a significantly increased preference for NADP. The catalytic efficiency of mutant RsPtxDHARRA for NADP (Kcat/KM)NADP was 44.1 μM–1 min–1, which was the highest among the previously reported phosphite dehydrogenases. Moreover, the RsPtxDHARRA mutant exhibited high thermostability at 45°C for up to 6 h and high tolerance to organic solvents, when bound with NADP. We also demonstrated the applicability of RsPtxDHARRA as an NADPH regeneration system in the coupled reaction of chiral conversion of 3-dehydroshikimate to shikimic acid by the thermophilic shikimate dehydrogenase of Thermus thermophilus HB8 at 45°C, which could not be supported by the parent RsPtxD enzyme. Therefore, the RsPtxDHARRA mutant might be a promising alternative NADPH regeneration system for practical applications.
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Bailleul, Gautier, Callum Robert Nicoll, Maria Laura Mascotti, Andrea Mattevi, and Marco W. Fraaije. "Ancestral reconstruction of mammalian FMO1 enables structural determination, revealing unique features that explain its catalytic properties." Journal of Biological Chemistry, December 21, 2020, jbc.RA120.016297. http://dx.doi.org/10.1074/jbc.ra120.016297.
Full textAbstract:
Mammals rely on the oxidative flavin-containing monooxygenases (FMOs) to detoxify numerous and potentially deleterious xenobiotics; this activity extends to many drugs, giving FMOs high pharmacological relevance. However, our knowledge regarding these membrane-bound enzymes has been greatly impeded by the lack of structural information. We anticipated that ancestral sequence recon¬struction could help us identify protein sequences that are more amenable to structural analysis. As such, we hereby reconstructed the mammalian ancestral protein sequences of both FMO1 and FMO4, denoted as AncFMO1 and AncFMO4, respectively. AncFMO1, sharing 89.5 % sequence identity with human FMO1, was successfully expressed as functional enzyme. It displayed typical FMO activity as demonstrated by oxygenating benzydamine, tamoxifen and thioanisole, drug-related compounds known to be also accepted by human FMO1, and both NADH and NADPH cofactors could act as electron donors, a feature only described for the FMO1 paralogs. AncFMO1 crystallized as a dimer and was structurally resolved at 3.0 Å resolution. The structure harbors typical FMO aspects with the FAD and NAD(P)H binding domains and a C-terminal transmembrane helix. Intriguingly, AncFMO1 also contains some unique features, including a significantly porous and exposed active site, and NADPH adopting a new conformation with the 2’-phosphate being pushed inside the NADP+ binding domain instead of being stretched out in the solvent. Overall, the ancestrally reconstructed mammalian AncFMO1 serves as the first structural model to corroborate and rationalize the catalytic properties of FMO1.
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Sriherfyna, Feronika Heppy, Minenosuke Matsutani, Kensuke Hirano, Hisashi Koike, Naoya Kataoka, Tetsuo Yamashita, Eiko Nakamaru-Ogiso, Kazunobu Matsushita, and Toshiharu Yakushi. "The Auxiliary NADH Dehydrogenase Plays a Crucial Role in Redox Homeostasis of Nicotinamide Cofactors in the Absence of the Periplasmic Oxidation System in Gluconobacter oxydans NBRC3293." Applied and Environmental Microbiology 87, no. 2 (October 30, 2020). http://dx.doi.org/10.1128/aem.02155-20.
Full textAbstract:
ABSTRACT Gluconobacter oxydans has the unique property of a glucose oxidation system in the periplasmic space, where glucose is oxidized incompletely to ketogluconic acids in a nicotinamide cofactor-independent manner. Elimination of the gdhM gene for membrane-bound glucose dehydrogenase, the first enzyme for the periplasmic glucose oxidation system, induces a metabolic change whereby glucose is oxidized in the cytoplasm to acetic acid. G. oxydans strain NBRC3293 possesses two molecular species of type II NADH dehydrogenase (NDH), the primary and auxiliary NDHs that oxidize NAD(P)H by reducing ubiquinone in the cell membrane. The substrate specificities of the two NDHs are different from each other: primary NDH (p-NDH) oxidizes NADH specifically but auxiliary NDH (a-NDH) oxidizes both NADH and NADPH. We constructed G. oxydans NBRC3293 derivatives defective in the ndhA gene for a-NDH, in the gdhM gene, and in both. Our ΔgdhM derivative yielded higher cell biomass on glucose, as reported previously, but grew at a lower rate than the wild-type strain. The ΔndhA derivative showed growth behavior on glucose similar to that of the wild type. The ΔgdhM ΔndhA double mutant showed greatly delayed growth on glucose, but its cell biomass was similar to that of the ΔgdhM strain. The double mutant accumulated intracellular levels of NAD(P)H and thus shifted the redox balance to reduction. Accumulated NAD(P)H levels might repress growth on glucose by limiting oxidative metabolisms in the cytoplasm. We suggest that a-NDH plays a crucial role in redox homeostasis of nicotinamide cofactors in the absence of the periplasmic oxidation system in G. oxydans. IMPORTANCE Nicotinamide cofactors NAD+ and NADP+ mediate redox reactions in metabolism. Gluconobacter oxydans, a member of the acetic acid bacteria, oxidizes glucose incompletely in the periplasmic space—outside the cell. This incomplete oxidation of glucose is independent of nicotinamide cofactors. However, if the periplasmic oxidation of glucose is abolished, the cells oxidize glucose in the cytoplasm by reducing nicotinamide cofactors. Reduced forms of nicotinamide cofactors are reoxidized by NADH dehydrogenase (NDH) on the cell membrane. We found that two kinds of NDH in G. oxydans have different substrate specificities: the primary enzyme is NADH specific, and the auxiliary one oxidizes both NADH and NADPH. Inactivation of the latter enzyme in G. oxydans cells in which we had induced cytoplasmic glucose oxidation resulted in elevated intracellular levels of NAD(P)H, limiting cell growth on glucose. We suggest that the auxiliary enzyme is important if G. oxydans grows independently of the periplasmic oxidation system.
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Yang, Ting, Ya-Hui Shao, Li-Zhong Guo, Xiang-Lin Meng, Hao Yu, and Wei-Dong Lu. "Role of N,N-Dimethylglycine and Its Catabolism to Sarcosine in Chromohalobacter salexigens DSM 3043." Applied and Environmental Microbiology 86, no. 17 (July 6, 2020). http://dx.doi.org/10.1128/aem.01186-20.
Full textAbstract:
ABSTRACT Chromohalobacter salexigens DSM 3043 can grow on N,N-dimethylglycine (DMG) as the sole C, N, and energy source and utilize sarcosine as the sole N source under aerobic conditions. However, little is known about the genes and enzymes involved in the conversion of DMG to sarcosine in this strain. In the present study, gene disruption and complementation assays indicated that the csal_0990, csal_0991, csal_0992, and csal_0993 genes are responsible for DMG degradation to sarcosine. The csal_0990 gene heterologously expressed in Escherichia coli was proven to encode an unusual DMG dehydrogenase (DMGDH). The enzyme, existing as a monomer of 79 kDa with a noncovalently bound flavin adenine dinucleotide, utilized both DMG and sarcosine as substrates and exhibited dual coenzyme specificity, preferring NAD+ to NADP+. The optimum pH and temperature of enzyme activity were determined to be 7.0 and 60°C, respectively. Kinetic parameters of the enzyme toward its substrates were determined accordingly. Under high-salinity conditions, the presence of DMG inhibited growth of the wild type and induced the production and accumulation of trehalose and glucosylglycerate intracellularly. Moreover, exogenous addition of DMG significantly improved the growth rates of the four DMG– mutants (Δcsal_0990, Δcsal_0991, Δcsal_0992, and Δcsal_0993) incubated at 37°C in S-M63 synthetic medium with sarcosine as the sole N source. 13C nuclear magnetic resonance (13C-NMR) experiments revealed that not only ectoine, glutamate, and N-acetyl-2,4-diaminobutyrate but also glycine betaine (GB), DMG, sarcosine, trehalose, and glucosylglycerate are accumulated intracellularly in the four mutants. IMPORTANCE Although N,N-dimethylglycine (DMG) dehydrogenase (DMGDH) activity was detected in cell extracts of microorganisms, the genes encoding microbial DMGDHs have not been determined until now. In addition, to our knowledge, the physiological role of DMG in moderate halophiles has never been investigated. In this study, we identified the genes involved in DMG degradation to sarcosine, characterized an unusual DMGDH, and investigated the role of DMG in Chromohalobacter salexigens DSM 3043 and its mutants. Our results suggested that the conversion of DMG to sarcosine is accompanied by intramolecular delivery of electrons in DMGDH and intermolecular electron transfer between DMGDH and other electron acceptors. Moreover, an unidentified methyltransferase catalyzing the production of glycine betaine (GB) from DMG but sharing no homology with the reported sarcosine DMG methyltransferases was predicted to be present in the cells. The results of this study expand our understanding of the physiological role of DMG and its catabolism to sarcosine in C. salexigens.