Tags

Type your tag names separated by a space and hit enter

A catalytic consensus motif for D-mannitol 2-dehydrogenase, a member of a polyol-specific long-chain dehydrogenase family, revealed by kinetic characterization of site-directed mutants of the enzyme from Pseudomonas fluorescens.
Biochem J. 2002 Oct 01; 367(Pt 1):13-8.BJ

Abstract

Lys-295, Asn-300 and His-303 of D-mannitol 2-dehydrogenase from Pseudomonas fluorescens were mutated individually into alanine (K295A, N300A and H303A respectively). Purified mutants displayed catalytic efficiencies for NAD(+)-dependent oxidation of D-mannitol 300-fold (H303A), 1000-fold (N300A) and approx. 400000-fold (K295A) below the wild-type level. Comparison of primary kinetic isotope effects on kinetic parameters for D-fructose reduction by wild-type and mutants at pH 10.0 demonstrate that Asn-300 has an auxiliary role in stabilization of the transition state of hydride transfer, and His-303 contributes to substrate positioning. The large solvent isotope effect of 11+/-1 on k (cat) for mannitol oxidation by K295A at pH((2)H) 10.5 suggests a role for Lys-295 in general base enzymic catalysis. Positional conservation of Lys-295, Asn-300 and His-303 across a family of polyol-specific long-chain dehydrogenases suggests a unique catalytic signature: Lys-Xaa(4)-Asn-Xaa(2)-His (where 'Xaa' denotes 'any amino acid').

Authors+Show Affiliations

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

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

12175334

Citation

Klimacek, Mario, and Bernd Nidetzky. "A Catalytic Consensus Motif for D-mannitol 2-dehydrogenase, a Member of a Polyol-specific Long-chain Dehydrogenase Family, Revealed By Kinetic Characterization of Site-directed Mutants of the Enzyme From Pseudomonas Fluorescens." The Biochemical Journal, vol. 367, no. Pt 1, 2002, pp. 13-8.
Klimacek M, Nidetzky B. A catalytic consensus motif for D-mannitol 2-dehydrogenase, a member of a polyol-specific long-chain dehydrogenase family, revealed by kinetic characterization of site-directed mutants of the enzyme from Pseudomonas fluorescens. Biochem J. 2002;367(Pt 1):13-8.
Klimacek, M., & Nidetzky, B. (2002). A catalytic consensus motif for D-mannitol 2-dehydrogenase, a member of a polyol-specific long-chain dehydrogenase family, revealed by kinetic characterization of site-directed mutants of the enzyme from Pseudomonas fluorescens. The Biochemical Journal, 367(Pt 1), 13-8.
Klimacek M, Nidetzky B. A Catalytic Consensus Motif for D-mannitol 2-dehydrogenase, a Member of a Polyol-specific Long-chain Dehydrogenase Family, Revealed By Kinetic Characterization of Site-directed Mutants of the Enzyme From Pseudomonas Fluorescens. Biochem J. 2002 Oct 1;367(Pt 1):13-8. PubMed PMID: 12175334.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A catalytic consensus motif for D-mannitol 2-dehydrogenase, a member of a polyol-specific long-chain dehydrogenase family, revealed by kinetic characterization of site-directed mutants of the enzyme from Pseudomonas fluorescens. AU - Klimacek,Mario, AU - Nidetzky,Bernd, PY - 2002/08/13/accepted PY - 2002/08/07/revised PY - 2002/06/17/received PY - 2002/8/15/pubmed PY - 2003/6/18/medline PY - 2002/8/15/entrez SP - 13 EP - 8 JF - The Biochemical journal JO - Biochem. J. VL - 367 IS - Pt 1 N2 - Lys-295, Asn-300 and His-303 of D-mannitol 2-dehydrogenase from Pseudomonas fluorescens were mutated individually into alanine (K295A, N300A and H303A respectively). Purified mutants displayed catalytic efficiencies for NAD(+)-dependent oxidation of D-mannitol 300-fold (H303A), 1000-fold (N300A) and approx. 400000-fold (K295A) below the wild-type level. Comparison of primary kinetic isotope effects on kinetic parameters for D-fructose reduction by wild-type and mutants at pH 10.0 demonstrate that Asn-300 has an auxiliary role in stabilization of the transition state of hydride transfer, and His-303 contributes to substrate positioning. The large solvent isotope effect of 11+/-1 on k (cat) for mannitol oxidation by K295A at pH((2)H) 10.5 suggests a role for Lys-295 in general base enzymic catalysis. Positional conservation of Lys-295, Asn-300 and His-303 across a family of polyol-specific long-chain dehydrogenases suggests a unique catalytic signature: Lys-Xaa(4)-Asn-Xaa(2)-His (where 'Xaa' denotes 'any amino acid'). SN - 0264-6021 UR - https://www.unboundmedicine.com/medline/citation/12175334/A_catalytic_consensus_motif_for_D_mannitol_2_dehydrogenase_a_member_of_a_polyol_specific_long_chain_dehydrogenase_family_revealed_by_kinetic_characterization_of_site_directed_mutants_of_the_enzyme_from_Pseudomonas_fluorescens_ L2 - https://portlandpress.com/biochemj/article-lookup/doi/10.1042/BJ20020932 DB - PRIME DP - Unbound Medicine ER -