PEDS Advance Access originally published online on July 12, 2005
Protein Engineering Design and Selection 2005 18(8):369-377; doi:10.1093/protein/gzi043
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A modified consensus approach to mutagenesis inverts the cofactor specificity of Bacillus stearothermophilus lactate dehydrogenase
1Instituto de Biotecnología/UNAM, Apartado Postal 510-3, Cuernavaca, Morelos, 62271, México and 2Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas at Austin, 1 University Station A4800, Austin, TX 78712, USA
3 To whom correspondence should be addressed. E-mail: andy.ellington{at}mail.utexas.edu
Lactate dehydrogenase from Bacillus stearothermophilus is specific for NAD+. There have been several attempts to alter the cofactor specificity of this enzyme, but these have yielded enzymes with relatively low activities that still largely prefer NAD+. A modified consensus approach was used to create a library of phylogenetically preferred amino acids situated near the cofactor binding site, and variants were screened for their ability to utilize NMN+. A triple mutant (Mut31) was discovered that proved to be more catalytically efficient than wild-type. Mut31 was also better at utilizing NAD+ than the wild-type enzyme and was weakly active with NADP+ and NMN+. An analysis of single amino acid substitutions suggested that all three mutations worked in a concerted fashion to yield robust cofactor utilization. When two previously identified amino acid substitutions were introduced into the Mut31 background, the resultant quintuply substituted enzyme not only utilized NADP+ far better than the wild-type enzyme, it actually inverted its preference for NAD+ and NADP+.
Keywords: cofactor specificity/consensus/directed evolution/lactate dehydrogenase/molecular evolution
Received October 28, 2004; revised May 12, 2005; accepted June 1, 2005.
Edited by Andrew Griffiths