Using directed evolution to probe the substrate specificity of mandelamide hydrolase.
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 k(cat)/K(m) for (R)- and (S)-mandelamide. This reduction in activity increased to six orders of magnitude for the G202V variant.
Wang
Pan-Fen
PF
College of Pharmacy, University of Michigan, 428 Church St, Ann Arbor, MI 48109, USA.
Yep
Alejandra
A
Kenyon
George L
GL
McLeish
Michael J
MJ
D000577:Amides / Q000378:metabolism; D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000641:Ammonia / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020134:Catalytic Domain; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D008333:Mandelic Acids / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015202:Protein Engineering; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D000577:Amides; D001426:Bacterial Proteins; D008333:Mandelic Acids; C010413:mandelamide; C013083:lactamide; D000641:Ammonia; D000581:Amidohydrolases; C037938:mandelic acid