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PEDS Advance Access published online on December 10, 2008
Protein Engineering Design and Selection, doi:10.1093/protein/gzn073
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Using directed evolution to probe the substrate specificity of mandelamide hydrolase

Pan-Fen Wang, Alejandra Yep, George L. Kenyon and Michael J. McLeish1,2

College of Pharmacy, University of Michigan, 428 Church St, Ann Arbor, MI 48109, USA

2 To whom correspondence should be addressed. E-mail: mcleish{at}iupui.edu

Mandelamide hydrolase (MAH), a member of the amidase signature family, catalyzes the hydrolysis of mandelamide to mandelate and ammonia. X-ray structures of several members of this family, but not that of MAH, have been reported. These reveal nearly superimposable conformations of the unusual Ser-cisSer-Lys catalytic triad. Conversely, the residues involved in substrate recognition are not conserved, implying that the binding pocket could be modified to change the substrate specificity, perhaps by directed evolution. Here we show that MAH is able to hydrolyze small aliphatic substrates such as lactamide, albeit with low efficiency. A selection method to monitor changes in mandelamide/lactamide preference was developed and used to identify several mutations affecting substrate binding. A homology model places some of these mutations close to the catalytic triad, presumably in the MAH active site. In particular, Gly202 appears to control the preference for aromatic substrates as the G202A variant showed three orders of magnitude decrease in kcat/Km for (R)- and (S)-mandelamide. This reduction in activity increased to six orders of magnitude for the G202V variant.

Keywords: enzyme catalysis/homology model/mutagenesis/protein engineering/selection

Received July 3, 2008; revised November 2, 2008; accepted November 7, 2008.

1 Present address: Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, 402 N. Blackford St, Indianapolis, IN 46202, USA.


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