Penicillin G acylase is an important enzyme in
the commercial production of semisynthetic penicillins
used to combat bacterial infections. Mutant strains of
Providencia rettgeri were generated from wild-type
cultures subjected to nutritional selective pressure. One
such mutant, Bro1, was able to use 6-bromohexanamide as
its sole nitrogen source. Penicillin acylase from the Bro1
strain exhibited an altered substrate specificity consistent
with the ability of the mutant to process 6-bromohexanamide.
The X-ray structure determination of this enzyme was undertaken
to understand its altered specificity and to help in the
design of site-directed mutants with desired specificities.
In this paper, the structure of the Bro1 penicillin G acylase
has been solved at 2.5 Å resolution by molecular
replacement. The R-factor after refinement is
0.154 and R-free is 0.165. Of the 758 residues
in the Bro1 penicillin acylase heterodimer (α-subunit,
205; β-subunit, 553), all but the eight C-terminal
residues of the α-subunit have been modeled based on
a partial Bro1 sequence and the complete wild-type P.
rettgeri sequence. A tightly bound calcium ion coordinated
by one residue from the α-subunit and five residues
from the β-subunit has been identified. This enzyme
belongs to the superfamily of Ntn hydrolases and uses Oγ
of Serβ1 as the characteristic N-terminal nucleophile.
A mutation of the wild-type Metα140 to Leu in the Bro1
acylase hydrophobic specificity pocket is evident from
the electron density and is consistent with the observed
specificity change for Bro1 acylase. The electron density
for the N-terminal Gln of the α-subunit is best modeled
by the cyclized pyroglutamate form. Examination of aligned
penicillin acylase and cephalosporin acylase primary sequences,
in conjunction with the P. rettgeri and Escherichia
coli penicillin acylase crystal structures, suggests
several mutations that could potentially allow penicillin
acylase to accept charged β-lactam R-groups and to
function as a cephalosporin acylase and thus be used in
the manufacture of semi-synthetic cephalosporins.