Structure of NADH-Dependent Carbonyl Reductase (CPCR2) from Candida parapsilosis Provides Insight into Mutations that Improve Catalytic Properties

Authors

  • Henry Man,

    1. York Structural Biology Laboratory, University of York, Heslington, York YO10 5DD (U.K.), Fax: (+44) 1904-328266
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  • Christoph Loderer,

    1. Department of Molecular Biotechnology, Institute of Microbiology, Technische Universität Dresden, 01062 Dresden (Germany), Fax: (+49) 351-463-39520
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  • Prof. Dr. Marion B. Ansorge-Schumacher,

    Corresponding author
    1. Department of Molecular Biotechnology, Institute of Microbiology, Technische Universität Dresden, 01062 Dresden (Germany), Fax: (+49) 351-463-39520
    • Marion B. Ansorge-Schumacher, Department of Molecular Biotechnology, Institute of Microbiology, Technische Universität Dresden, 01062 Dresden (Germany), Fax: (+49) 351-463-39520

      Gideon Grogan, York Structural Biology Laboratory, University of York, Heslington, York YO10 5DD (U.K.), Fax: (+44) 1904-328266

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  • Dr. Gideon Grogan

    Corresponding author
    1. York Structural Biology Laboratory, University of York, Heslington, York YO10 5DD (U.K.), Fax: (+44) 1904-328266
    • Marion B. Ansorge-Schumacher, Department of Molecular Biotechnology, Institute of Microbiology, Technische Universität Dresden, 01062 Dresden (Germany), Fax: (+49) 351-463-39520

      Gideon Grogan, York Structural Biology Laboratory, University of York, Heslington, York YO10 5DD (U.K.), Fax: (+44) 1904-328266

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Abstract

The (S)-selective carbonyl reductase CPCR2 from Candida parapsilosis is a member of the medium-chain reductase family of enzymes and is a useful biocatalyst for the reduction of prochiral ketone substrates. The structure of CPCR2 was determined in complex with the cofactor NADH [NADH=reduced form of nicotinamide adenine dinucleotide (NAD+)] to a resolution of 2.05 Å. Two dimers formed a tetramer in the asymmetric unit, but solution studies confirmed that a dimer was the predominant species in solution. In the monomer, the NADH cofactor is bound at the interface between the nucleotide binding domain and the catalytic domain, and the Re-face hydride of the nicotinamide ring is presented to a hydrophobic binding pocket featuring the Leu262, Phe285, Trp286, Trp116, Leu119, Leu55 and Val50 residues, which leads to the surface of the enzyme. The catalytic zinc and coordinating amino acid side chains were observed in different conformations in the different monomers. In three out of four monomers, the zinc was coordinated by His65, Asp154, Glu66 and a water molecule; in the other subunit, an alternative coordination sphere, consisting of His65, Asp154, Cys44 and a water molecule, was observed. The change in coordination was accompanied by a movement of a mobile region of the protein chain between residues 43 and 63, which bears Cys44. The structure of CPCR2 provides further evidence of a dynamic coordination sphere for zinc in medium-chain reductase dependent catalysis. It also sheds light on previous engineering studies on CPCR2 that were performed in the absence of structural data and provides a robust and reliable new model for further experiments directed towards improvement or alteration of CPCR2 activity.

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