PEDS Advance Access originally published online on July 14, 2004
Protein Engineering Design and Selection 2004 17(5):473-480; doi:10.1093/protein/gzh057
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Structural and kinetic studies on ligand binding in wild-type and active-site mutants of penicillin acylase
1Department of Biochemistry and 2BIOSON Research Institute, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
3 To whom correspondence should be addressed. E-mail: d.b.janssen{at}chem.rug.nl
Penicillin acylase catalyses the condensation of C
-substituted phenylacetic acids with ß-lactam nucleophiles, producing semi-synthetic ß-lactam antibiotics. For efficient synthesis a low affinity for phenylacetic acid and a high affinity for C-substituted phenylacetic acid derivatives is desirable. We made three active site mutants, F146Y, ßF24A and F146Y/ßF24A, which all had a 2- to 10-fold higher affinity for C-substituted compounds than wild-type enzyme. In addition, ßF24A had a 20-fold reduced affinity for phenylacetic acid. The molecular basis of the improved properties was investigated by X-ray crystallography. These studies showed that the higher affinity of F146Y for (R)--methylphenylacetic acid can be explained by van der Waals interactions between Y146:OH and the C-substituent. The ßF24A mutation causes an opening of the phenylacetic acid binding site. Only (R)--methylphenylacetic acid, but not phenylacetic acid, induces a conformation with the ligand tightly bound, explaining the weak binding of phenylacetic acid. A comparison of the ßF24A structure with other open conformations of penicillin acylase showed that ßF24 has a fixed position, whereas F146 acts as a flexible lid on the binding site and reorients its position to achieve optimal substrate binding.
Received March 30, 2004; revised June 18, 2004; accepted June 26, 2004.
Edited by Mirek Cygler