Characterization of recombinant xylitol dehydrogenase from Galactocandida mastotermitis expressed in Escherichia coli

Nidetzky, Bernd; Helmer, Heidemarie; Klimacek, Mario; Lunzer, Regina; Mayer, Gerhard

doi:10.1016/s0009-2797(02)00215-6

Cited by 27 publications

(16 citation statements)

References 22 publications

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“…Therefore, the pro-R hydrogen points towards Tyr94, indicating A-face 4-pro-R hydride transfer at the prochiral center of NADPH to the β-carbon of the double bond of the substrate, as has been observed experimentally also for some other family members. 39,40 The modeling calculations also show that the conformation of TYGGM-(β5-β#) loop changes, causing a widening of the pantetheine binding cleft. This suggests that the NADPH binds in the active site of MECR/ETR1 prior to the fatty acyl thioester substrate.…”

Section: Discussionmentioning

confidence: 93%

Structural Enzymological Studies of 2-Enoyl Thioester Reductase of the Human Mitochondrial FAS II Pathway: New Insights into Its Substrate Recognition Properties

Chen

Pudas

Sharma

et al. 2008

Journal of Molecular Biology

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Section: Discussionmentioning

confidence: 93%

Structural Enzymological Studies of 2-Enoyl Thioester Reductase of the Human Mitochondrial FAS II Pathway: New Insights into Its Substrate Recognition Properties

Chen

Pudas

Sharma

et al. 2008

Journal of Molecular Biology

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“…Other examples exist where dehydrogenases are preferentially, but not completely ordered (e.g. d-xylitol dehydrogenase [40]). Finally, the D V ⁄ K Pt values for PTDH-E175A ⁄ A176R were close to those for the WT enzyme for both cofactors ( Table 2).…”

Section: Resultsmentioning

confidence: 99%

Mechanistic investigation of a highly active phosphite dehydrogenase mutant and its application for NADPH regeneration

2005

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NAD(P)H regeneration is important for biocatalytic reactions that require these costly cofactors. A mutant phosphite dehydrogenase (PTDH‐E175A/A176R) that utilizes both NAD and NADP efficiently is a very promising system for NAD(P)H regeneration. In this work, both the kinetic mechanism and practical application of PTDH‐E175A/A176R were investigated for better understanding of the enzyme and to provide a basis for future optimization. Kinetic isotope effect studies with PTDH‐E175A/A176R showed that the hydride transfer step is (partially) rate determining with both NAD and NADP giving DV values of 2.2 and 1.7, respectively, and DV/Km,phosphite values of 1.9 and 1.7, respectively. To better comprehend the relaxed cofactor specificity, the cofactor dissociation constants were determined utilizing tryptophan intrinsic fluorescence quenching. The dissociation constants of NAD and NADP with PTDH‐E175A/A176R were 53 and 1.9 µm, respectively, while those of the products NADH and NADPH were 17.4 and 1.22 µm, respectively. Using sulfite as a substrate mimic, the binding order was established, with the cofactor binding first and sulfite binding second. The low dissociation constant for the cofactor product NADPH combined with the reduced values for DV and kcat implies that product release may become partially rate determining. However, product inhibition does not prevent efficient in situ NADPH regeneration by PTDH‐E175A/A176R in a model system in which xylose was converted into xylitol by NADP‐dependent xylose reductase. The in situ regeneration proceeded at a rate approximately fourfold faster with PTDH‐E175A/A176R than with either WT PTDH or a NADP‐specific Pseudomonas sp.101 formate dehydrogenase mutant with a total turnover number for NADPH of 2500.

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“…Unless mentioned otherwise, all materials and chemicals have been described elsewhere [18]. 2 H 2 O solvent (99.9 % 2 H) and Dsorbitol were from Sigma-Aldrich.…”

Section: Methodsmentioning

confidence: 99%

Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu154→Cys forms of yeast xylitol dehydrogenase

Klimacek

Hellmer

Nidetzky

2007

Biochemical Journal

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Co-ordination of catalytic Zn2+ in sorbitol/xylitol dehydrogenases of the medium-chain dehydrogenase/reductase superfamily involves direct or water-mediated interactions from a glutamic acid residue, which substitutes a homologous cysteine ligand in alcohol dehydrogenases of the yeast and liver type. Glu154 of xylitol dehydrogenase from the yeast Galactocandida mastotermitis (termed GmXDH) was mutated to a cysteine residue (E154C) to revert this replacement. In spite of their variable Zn2+ content (0.10-0.40 atom/subunit), purified preparations of E154C exhibited a constant catalytic Zn2+ centre activity (kcat) of 1.19+/-0.03 s(-1) and did not require exogenous Zn2+ for activity or stability. E154C retained 0.019+/-0.003% and 0.74+/-0.03% of wild-type catalytic efficiency (kcat/K(sorbitol)=7800+/-700 M(-1) x s(-1)) and kcat (=161+/-4 s(-1)) for NAD+-dependent oxidation of sorbitol at 25 degrees C respectively. The pH profile of kcat/K(sorbitol) for E154C decreased below an apparent pK of 9.1+/-0.3, reflecting a shift in pK by about +1.7-1.9 pH units compared with the corresponding pH profiles for GmXDH and sheep liver sorbitol dehydrogenase (termed slSDH). The difference in pK for profiles determined in 1H2O and 2H2O solvent was similar and unusually small for all three enzymes (approximately +0.2 log units), suggesting that the observed pK in the binary enzyme-NAD+ complexes could be due to Zn2+-bound water. Under conditions eliminating their different pH-dependences, wild-type and mutant GmXDH displayed similar primary and solvent deuterium kinetic isotope effects of 1.7+/-0.2 (E154C, 1.7+/-0.1) and 1.9+/-0.3 (E154C, 2.4+/-0.2) on kcat/K(sorbitol) respectively. Transient kinetic studies of NAD+ reduction and proton release during sorbitol oxidation by slSDH at pH 8.2 show that two protons are lost with a rate constant of 687+/-12 s(-1) in the pre-steady state, which features a turnover of 0.9+/-0.1 enzyme equivalents as NADH was produced with a rate constant of 409+/-3 s(-1). The results support an auxiliary participation of Glu154 in catalysis, and possible mechanisms of proton transfer in sorbitol/xylitol dehydrogenases are discussed.

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Characterization of recombinant xylitol dehydrogenase from Galactocandida mastotermitis expressed in Escherichia coli

Cited by 27 publications

References 22 publications

Structural Enzymological Studies of 2-Enoyl Thioester Reductase of the Human Mitochondrial FAS II Pathway: New Insights into Its Substrate Recognition Properties

Structural Enzymological Studies of 2-Enoyl Thioester Reductase of the Human Mitochondrial FAS II Pathway: New Insights into Its Substrate Recognition Properties

Mechanistic investigation of a highly active phosphite dehydrogenase mutant and its application for NADPH regeneration

Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu154→Cys forms of yeast xylitol dehydrogenase

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