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

• Published in: ChemCatChem Vol. 6; no. 4; pp. 1103 - 1111
• Main Authors: Man, Henry, Loderer, Christoph, Ansorge-Schumacher, Marion B., Grogan, Gideon
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 01.04.2014; WILEY‐VCH Verlag; Wiley Subscription Services, Inc
• Subjects: alcohol dehydrogenase; Binding; carbonyl reductase; Carbonyls; Catalysis; Chains; Dimers; enzymes; ketoreductase; Molecular conformation; Monomers; Mutation; Nicotinamide; Nicotinamide adenine dinucleotide; Proteins; reduction; Residues; Substrates; Water chemistry; Zinc coordination
• ISSN: 1867-3880; 1867-3899
• DOI: 10.1002/cctc.201300788

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. Mutant ninja enzymes: The structure of the carbonyl reductase CPCR2 reveals the context of the mutational sites that have led to its improved performance, including mutations within the active‐site channel that affect substrate recognition and those at the dimer interface that improve stability. The structure also provides further evidence for the dynamic coordination of catalytic zinc within the active site of the medium‐chain reductase family of enzymes.