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PEDS Advance Access published online on July 12, 2005
Protein Engineering Design and Selection, doi:10.1093/protein/gzi043
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oupjournals.org
Received October 28, 2004
Revised May 12, 2005
Accepted June 1, 2005

Article

A modified consensus approach to mutagenesis inverts the cofactor specificity of Bacillus stearothermophilus lactate dehydrogenase

Humberto Flores 1 and Andrew D. Ellington 2*

1 Instituto de Biotecnología/UNAM, Apartado Postal 510-3, Cuernavaca, Morelos, 62271, México
2 Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas at Austin, 1 University Station A4800, Austin, TX 78712, USA

* To whom correspondence should be addressed.
Andrew D. Ellington, E-mail: andy.ellington{at}mail.utexas.edu


  Abstract

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.
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