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Penicillin acylase (penicillin amidohydrolase, EC 3.5.1.11) is widely
distributed among microorganisms, including bacteria, yeast and filamentous
fungi. It is used on an industrial scale for the production of
6-aminopenicillanic acid, the starting material for the synthesis of
semi-synthetic penicillins. Its in vivo role remains unclear, however, and the
observation that expression of the Escherichia coli enzyme in vivo is regulated
by both temperature and phenylacetic acid has prompted speculation that the
enzyme could be involved in the assimilation of aromatic compounds as carbon
sources in the organism's free-living mode. The mature E. coli enzyme is a
periplasmic 80K heterodimer of A and B chains (209 and 566 amino acids,
respectively) synthesized as a single cytoplasmic precursor containing a
26-amino-acid signal sequence to direct export to the cytoplasm and a
54-amino-acid spacer between the A and B chains which may influence the final
folding of the chains. The N-terminal serine of the B chain reacts with
phenylmethylsulphonyl fluoride, which is consistent with a catalytic role for
the serine hydroxyl group. Modifying this serine to a cysteine inactivates the
enzyme, whereas threonine, arginine or glycine substitution prevents in vivo
processing of the enzyme, indicating that this must be an important recognition
site for cleavage. Here we report the crystal structure of penicillin acylase at
1.9 A resolution. Our analysis shows that the environment of the catalytically
active N-terminal serine of the B chain contains no adjacent histidine
equivalent to that found in the serine proteases. The nearest base to the
hydroxyl of this serine is its own alpha-amino group, which may act by a new
mechanism to endow the enzyme with its catalytic properties.
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