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PEDS Advance Access published online on July 14, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh057
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Received March 30, 2004
Revised June 18, 2004
Accepted June 26, 2004

Article

Structural and kinetic studies on ligand binding in wild-type and active-site mutants of penicillin acylase

Wynand B. L. Alkema 1, Charles M. H. Hensgens 2, Harm J. Snijder 2, Evelien Keizer 2, Bauke W. Dijkstra 2, Dick B. Janssen 1*

1 Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
2 BIOSON Research Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands; Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands

* To whom correspondence should be addressed. E-mail: d.b.janssen{at}chem.rug.nl.


  Abstract

Penicillin acylase catalyses the condensation of C{alpha}-substituted phenylacetic acids with {beta}-lactam nucleophiles, producing semi-synthetic {beta}-lactam antibiotics. For efficient synthesis a low affinity for phenylacetic acid and a high affinity for C{alpha}-substituted phenylacetic acid derivatives is desirable. We made three active site mutants, {alpha}F146Y, {beta}F24A and {alpha}F146Y/{beta}F24A, which all had a 2- to 10-fold higher affinity for C{alpha}-substituted compounds than wild-type enzyme. In addition, {beta}F24A 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 {alpha}F146Y for (R)-{alpha}-methylphenylacetic can be explained by Van der Waals interactions between {alpha}Y146:OH and the C{alpha}-substituent. The {beta}F24A mutation causes an opening of the phenylacetic acid binding site. Only acid, but not phenylacetic acid, induces a conformation with the ligand tightly bound, explaining the weak binding of phenylacetic acid. A comparison of the {beta}F24A structure with other open conformations of penicillin acylase showed that {beta}F24 has a fixed position, whereas {alpha}F146 acts as a flexible lid on the binding site and reorients its position to achieve optimal substrate binding.

Keywords: penicillin acylase; mutants; ligand binding; 3D structure; kinetics.
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