1KEC: Penicillin Acylase Mutant With Phenyl Proprionic Acid

Citation:
Abstract
Penicillin acylase catalyses the condensation of Calpha-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 Calpha-substituted phenylacetic acid derivatives is desirable. We made three active site mutants, alphaF146Y, betaF24A and alphaF146Y/betaF24A, which all had a 2- to 10-fold higher affinity for Calpha-substituted compounds than wild-type enzyme. In addition, betaF24A 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 alphaF146Y for (R)-alpha-methylphenylacetic acid can be explained by van der Waals interactions between alphaY146:OH and the Calpha-substituent. The betaF24A mutation causes an opening of the phenylacetic acid binding site. Only (R)-alpha-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 betaF24A structure with other open conformations of penicillin acylase showed that betaF24 has a fixed position, whereas alphaF146 acts as a flexible lid on the binding site and reorients its position to achieve optimal substrate binding.
PDB ID: 1KECDownload
MMDB ID: 23851
PDB Deposition Date: 2001/11/15
Updated in MMDB: 2012/12
Experimental Method:
x-ray diffraction
Resolution: 2.3  Å
Source Organism:
Similar Structures:
Biological Unit for 1KEC: dimeric; determined by author and by software (PISA)
Molecular Components in 1KEC
Label Count Molecule
Proteins (2 molecules)
1
Penicillin Acylase Alpha Subunit
Molecule annotation
1
Penicillin Acylase Beta Subunit
Molecule annotation
Chemicals (2 molecules)
1
1
2
1
* Click molecule labels to explore molecular sequence information.

Citing MMDB
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