Crystal Structure of a
Preacylation Complex of the
β‑Lactamase Inhibitor Sulbactam Bound to a Sulfenamide
Bond-Containing Thiol-β-lactamase
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Abstract
The rise of inhibitor-resistant and other β-lactamase
variants
is generating an interest in developing new β-lactamase
inhibitors to complement currently available antibiotics. To gain
insight into the chemistry of inhibitor recognition, we determined
the crystal structure of the inhibitor preacylation complex of sulbactam,
a clinical β-lactamase inhibitor, bound in the active site of
the S70C variant of SHV-1 β-lactamase, a resistance enzyme that
is normally present in <i>Klebsiella pneumoniae</i>. The
S70C mutation was designed to affect the reactivity of that catalytic
residue to allow for capture of the preacylation complex. Unexpectedly,
the 1.45 Å resolution inhibitor complex structure revealed that
residue C70 is involved in a sulfenamide bond with K73. Such a covalent
bond is not present in the wild-type SHV-1 or in an apo S70C structure
also determined in this study. This bond likely contributed significantly
to obtaining the preacylation complex with sulbactam due to further
decreased reactivity toward substrates. The intact sulbactam is positioned
in the active site such that its carboxyl moiety interacts with R244,
S130, and T235 and its carbonyl moiety is situated in the oxyanion
hole. To our knowledge, in addition to being the first preacylation
inhibitor β-lactamase complex, this is also the first observation
of a sulfenamide bond between a cysteine and lysine in an active site.
Not only could our results aid, therefore, structure-based inhibitor
design efforts in class A β-lactamases, but the sulfenamide-bond
forming approach to yield preacylation complexes could also be applied
to other classes of β-lactamases and penicillin-binding proteins
with the SXXK motif