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From alcohol dehydrogenase to a "one-way" carbonyl reductase by active-site redesign: a mechanistic study of mannitol 2-dehydrogenase from pseudomonas fluorescens.
J Biol Chem. 2010 Oct 01; 285(40):30644-53.JB

Abstract

Directional preference in catalysis is often used to distinguish alcohol dehydrogenases from carbonyl reductases. However, the mechanistic basis underpinning this discrimination is weak. In mannitol 2-dehydrogenase from Pseudomonas fluorescens, stabilization of (partial) negative charge on the substrate oxyanion by the side chains of Asn-191 and Asn-300 is a key feature of catalysis in the direction of alcohol oxidation. We have disrupted this ability through individual and combined substitutions of the two asparagines by aspartic acid. Kinetic data and their thermodynamic analysis show that the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD(+)-mannitol (K(int)) was altered dramatically (10(4)- to 10(5)-fold) from being balanced in the wild-type enzyme (K(int) ≈ 3) to favoring enzyme-NAD(+)-mannitol in the single site mutants, N191D and N300D. The change in K(int) reflects a selective slowing down of the mannitol oxidation rate, resulting because Asn --> Asp replacement (i) disfavors partial abstraction of alcohol proton by Lys-295 in a step preceding catalytic hydride transfer, and (ii) causes stabilization of a nonproductive enzyme-NAD(+)-mannitol complex. N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pK values of 5.3 ± 0.1 and 6.3 ± 0.2, respectively. The mutant incorporating both Asn-->Asp substitutions behaved as a slow "fructose reductase" at pH 5.2, lacking measurable activity for mannitol oxidation in the pH range 6.8-10. A mechanism is suggested in which polarization of the substrate carbonyl by a doubly protonated diad of Asp and Lys-295 facilitates NADH-dependent reduction of fructose by N191D and N300D under optimum pH conditions. Creation of an effectively "one-way" reductase by active-site redesign of a parent dehydrogenase has not been previously reported and holds promise in the development of carbonyl reductases for application in organic synthesis.

Authors+Show Affiliations

Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria.No affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

20639204

Citation

Klimacek, Mario, and Bernd Nidetzky. "From Alcohol Dehydrogenase to a "one-way" Carbonyl Reductase By Active-site Redesign: a Mechanistic Study of Mannitol 2-dehydrogenase From Pseudomonas Fluorescens." The Journal of Biological Chemistry, vol. 285, no. 40, 2010, pp. 30644-53.
Klimacek M, Nidetzky B. From alcohol dehydrogenase to a "one-way" carbonyl reductase by active-site redesign: a mechanistic study of mannitol 2-dehydrogenase from pseudomonas fluorescens. J Biol Chem. 2010;285(40):30644-53.
Klimacek, M., & Nidetzky, B. (2010). From alcohol dehydrogenase to a "one-way" carbonyl reductase by active-site redesign: a mechanistic study of mannitol 2-dehydrogenase from pseudomonas fluorescens. The Journal of Biological Chemistry, 285(40), 30644-53. https://doi.org/10.1074/jbc.M110.108993
Klimacek M, Nidetzky B. From Alcohol Dehydrogenase to a "one-way" Carbonyl Reductase By Active-site Redesign: a Mechanistic Study of Mannitol 2-dehydrogenase From Pseudomonas Fluorescens. J Biol Chem. 2010 Oct 1;285(40):30644-53. PubMed PMID: 20639204.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - From alcohol dehydrogenase to a "one-way" carbonyl reductase by active-site redesign: a mechanistic study of mannitol 2-dehydrogenase from pseudomonas fluorescens. AU - Klimacek,Mario, AU - Nidetzky,Bernd, Y1 - 2010/07/16/ PY - 2010/7/20/entrez PY - 2010/7/20/pubmed PY - 2010/10/27/medline SP - 30644 EP - 53 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 285 IS - 40 N2 - Directional preference in catalysis is often used to distinguish alcohol dehydrogenases from carbonyl reductases. However, the mechanistic basis underpinning this discrimination is weak. In mannitol 2-dehydrogenase from Pseudomonas fluorescens, stabilization of (partial) negative charge on the substrate oxyanion by the side chains of Asn-191 and Asn-300 is a key feature of catalysis in the direction of alcohol oxidation. We have disrupted this ability through individual and combined substitutions of the two asparagines by aspartic acid. Kinetic data and their thermodynamic analysis show that the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD(+)-mannitol (K(int)) was altered dramatically (10(4)- to 10(5)-fold) from being balanced in the wild-type enzyme (K(int) ≈ 3) to favoring enzyme-NAD(+)-mannitol in the single site mutants, N191D and N300D. The change in K(int) reflects a selective slowing down of the mannitol oxidation rate, resulting because Asn --> Asp replacement (i) disfavors partial abstraction of alcohol proton by Lys-295 in a step preceding catalytic hydride transfer, and (ii) causes stabilization of a nonproductive enzyme-NAD(+)-mannitol complex. N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pK values of 5.3 ± 0.1 and 6.3 ± 0.2, respectively. The mutant incorporating both Asn-->Asp substitutions behaved as a slow "fructose reductase" at pH 5.2, lacking measurable activity for mannitol oxidation in the pH range 6.8-10. A mechanism is suggested in which polarization of the substrate carbonyl by a doubly protonated diad of Asp and Lys-295 facilitates NADH-dependent reduction of fructose by N191D and N300D under optimum pH conditions. Creation of an effectively "one-way" reductase by active-site redesign of a parent dehydrogenase has not been previously reported and holds promise in the development of carbonyl reductases for application in organic synthesis. SN - 1083-351X UR - https://www.unboundmedicine.com/medline/citation/20639204/From_alcohol_dehydrogenase_to_a_"one_way"_carbonyl_reductase_by_active_site_redesign:_a_mechanistic_study_of_mannitol_2_dehydrogenase_from_pseudomonas_fluorescens_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=20639204 DB - PRIME DP - Unbound Medicine ER -