Medline/PubMed Citation | Related Articles in PubMed | Download to Citation Matcher

Biochem. J. (2002) 367, (841–847) (Printed in Great Britain)

Engineering of coenzyme specificity of formate dehydrogenase from Saccharomyces cerevisiae

Alexander E. SEROV , Anna S. POPOVA , Vladimir V. FEDORCHUK and Vladimir I. TISHKOV1

Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Leninskie Gory, 119992 Moscow, Russian Federation

Key words: cofactor preference, mutagenesis, Pseudomonas sp.101, substrate binding, three-dimensional structure modelling.

Abbreviations used: D196A, Asp¹⁹⁶Ala; D195S, Asp¹⁹⁵Ser; FDH, formate dehydrogenase; CmeFDH, Candida methylica FDH; PseFDH, Pseudomonas sp.101 FDH; SceFDH, Saccharomyces cerevisiae FDH; Y197R, Tyr¹⁹⁷Arg.

¹To whom correspondence should be addressed (e-mail vit@enz.chem.msu.ru).

A eukaryotic formate dehydrogenase (EC 1.2.1.2, FDH) with its substrate specificity changed from NAD⁺ to NADP⁺ has been constructed by introducing two single-point mutations, Asp¹⁹⁶Ala (D196A) and Tyr¹⁹⁷Arg (Y197R). The mutagenesis was based on the results of homology modelling of a NAD⁺-specific FDH from Saccharomyces cerevisiae (SceFDH) using the Pseudomonas sp.101 FDH (PseFDH) crystal structure as a template. The resulting model structure suggested that Asp¹⁹⁶ and Tyr¹⁹⁷ mediate the absolute coenzyme specificity of SceFDH for NAD⁺.

Received 5 March 2002/22 July 2002; accepted 29 July 2002

Published as BJ Immediate Publication 29 July 2002, DOI 10.1042/BJ20020379

The Biochemical Society, London © 2002