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The oxyanion hole of Pseudomonas fluorescens mannitol 2-dehydrogenase: a novel structural motif for electrostatic stabilization in alcohol dehydrogenase active sites.
Biochem J. 2009 Dec 23; 425(2):455-63.BJ

Abstract

The side chains of Asn191 and Asn300 constitute a characteristic structural motif of the active site of Pseudomonas fluorescens mannitol 2-dehydrogenase that lacks precedent in known alcohol dehydrogenases and resembles the canonical oxyanion binding pocket of serine proteases. We have used steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in substrates and solvent on the enzymatic rates to delineate catalytic consequences resulting from individual and combined replacements of the two asparagine residues by alanine. The rate constants for the overall hydride transfer to and from C-2 of mannitol, which were estimated as approximately 5 x 102 s-1 and approximately 1.5 x 103 s-1 in the wild-type enzyme respectively, were selectively slowed, between 540- and 2700-fold, in single-site mannitol 2-dehydrogenase mutants. These effects were additive in the corresponding doubly mutated enzyme, suggesting independent functioning of the two asparagine residues in catalysis. Partial disruption of the oxyanion hole in single-site mutants caused an upshift, by >or=1.2 pH units, in the kinetic pK of the catalytic acid-base Lys295 in the enzyme-NAD+-mannitol complex. The oxyanion hole of mannitol 2-dehydrogenase is suggested to drive a precatalytic conformational equilibrium at the ternary complex level in which the reactive group of the substrate is 'activated' for chemical conversion through its precise alignment with the unprotonated side chain of Lys295 (mannitol oxidation) and C=O bond polarization by the carboxamide moieties of Asn191 and Asn300 (fructose reduction). In the subsequent hydride transfer step, the two asparagine residues provide approximately 40 kJ/mol of electrostatic stabilization.

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

19857201

Citation

Klimacek, Mario, and Bernd Nidetzky. "The Oxyanion Hole of Pseudomonas Fluorescens Mannitol 2-dehydrogenase: a Novel Structural Motif for Electrostatic Stabilization in Alcohol Dehydrogenase Active Sites." The Biochemical Journal, vol. 425, no. 2, 2009, pp. 455-63.
Klimacek M, Nidetzky B. The oxyanion hole of Pseudomonas fluorescens mannitol 2-dehydrogenase: a novel structural motif for electrostatic stabilization in alcohol dehydrogenase active sites. Biochem J. 2009;425(2):455-63.
Klimacek, M., & Nidetzky, B. (2009). The oxyanion hole of Pseudomonas fluorescens mannitol 2-dehydrogenase: a novel structural motif for electrostatic stabilization in alcohol dehydrogenase active sites. The Biochemical Journal, 425(2), 455-63. https://doi.org/10.1042/BJ20091441
Klimacek M, Nidetzky B. The Oxyanion Hole of Pseudomonas Fluorescens Mannitol 2-dehydrogenase: a Novel Structural Motif for Electrostatic Stabilization in Alcohol Dehydrogenase Active Sites. Biochem J. 2009 Dec 23;425(2):455-63. PubMed PMID: 19857201.
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TY - JOUR T1 - The oxyanion hole of Pseudomonas fluorescens mannitol 2-dehydrogenase: a novel structural motif for electrostatic stabilization in alcohol dehydrogenase active sites. AU - Klimacek,Mario, AU - Nidetzky,Bernd, Y1 - 2009/12/23/ PY - 2009/10/28/entrez PY - 2009/10/28/pubmed PY - 2010/1/15/medline SP - 455 EP - 63 JF - The Biochemical journal JO - Biochem. J. VL - 425 IS - 2 N2 - The side chains of Asn191 and Asn300 constitute a characteristic structural motif of the active site of Pseudomonas fluorescens mannitol 2-dehydrogenase that lacks precedent in known alcohol dehydrogenases and resembles the canonical oxyanion binding pocket of serine proteases. We have used steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in substrates and solvent on the enzymatic rates to delineate catalytic consequences resulting from individual and combined replacements of the two asparagine residues by alanine. The rate constants for the overall hydride transfer to and from C-2 of mannitol, which were estimated as approximately 5 x 102 s-1 and approximately 1.5 x 103 s-1 in the wild-type enzyme respectively, were selectively slowed, between 540- and 2700-fold, in single-site mannitol 2-dehydrogenase mutants. These effects were additive in the corresponding doubly mutated enzyme, suggesting independent functioning of the two asparagine residues in catalysis. Partial disruption of the oxyanion hole in single-site mutants caused an upshift, by >or=1.2 pH units, in the kinetic pK of the catalytic acid-base Lys295 in the enzyme-NAD+-mannitol complex. The oxyanion hole of mannitol 2-dehydrogenase is suggested to drive a precatalytic conformational equilibrium at the ternary complex level in which the reactive group of the substrate is 'activated' for chemical conversion through its precise alignment with the unprotonated side chain of Lys295 (mannitol oxidation) and C=O bond polarization by the carboxamide moieties of Asn191 and Asn300 (fructose reduction). In the subsequent hydride transfer step, the two asparagine residues provide approximately 40 kJ/mol of electrostatic stabilization. SN - 1470-8728 UR - https://www.unboundmedicine.com/medline/citation/19857201/The_oxyanion_hole_of_Pseudomonas_fluorescens_mannitol_2_dehydrogenase:_a_novel_structural_motif_for_electrostatic_stabilization_in_alcohol_dehydrogenase_active_sites_ L2 - https://portlandpress.com/biochemj/article-lookup/doi/10.1042/BJ20091441 DB - PRIME DP - Unbound Medicine ER -