1K7D

Penicillin Acylase with Phenyl Proprionic Acid

Summary for 1K7D

• Entry DOI: 10.2210/pdb1k7d/pdb
• Related: 1JX9 1K5Q 1K5S 1PNK
• Descriptor: Penicillin Acylase alpha subunit, penicillin Acylase beta subunit, CALCIUM ION, ... (5 entities in total)
• Functional Keywords: ntn-hydrolase fold, helices, beta-strands, phenyl proprionic acid, hydrolase
• Biological source: Escherichia coli; More
• Total number of polymer chains: 2
• Total formula weight: 86458.57

Authors

Hensgens, C.M.H.,Keizer, E.,Snijder, H.J.,Dijkstra, B.W. (deposition date: 2001-10-19, release date: 2003-09-02, Last modification date: 2024-12-25)

Primary citation

Alkema, W.B.L.,Hensgens, C.M.H.,Snijder, H.J.,Keizer, E.,Dijkstra, B.W.,Janssen, D.B.
Structural and kinetic studies on ligand binding in wild-type and active-site mutants of penicillin acylase.
Protein Eng.Des.Sel., 17:473-480, 2004

PubMed 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.

PubMed: 15254299
DOI: 10.1093/protein/gzh057

Experimental method

X-RAY DIFFRACTION (2.15 Å)