Structure of beta-lactam synthetase reveals how to synthesize antibiotics instead of asparagine.
The enzyme beta-lactam synthetase (beta-LS) catalyzes the formation of the beta-lactam ring in clavulanic acid, a clinically important beta-lactamase inhibitor. Whereas the penicillin beta-lactam ring is generated by isopenicillin N synthase (IPNS) in the presence of ferrous ion and dioxygen, beta-LS uses ATP and Mg2+ as cofactors. According to sequence alignments, beta-LS is homologous to class B asparagine synthetases (AS-Bs), ATP/Mg2+-dependent enzymes that convert aspartic acid to asparagine. Here we report the first crystal structure of a beta-LS. The 1.95 A resolution structure of Streptomyces clavuligerus beta-LS provides a fully resolved view of the active site in which substrate, closely related ATP analog alpha,beta-methyleneadenosine 5'-triphosphate (AMP-CPP) and a single Mg2+ ion are present. A high degree of substrate preorganization is observed. Comparison to Escherichia coli AS-B reveals the evolutionary changes that have taken place in beta-LS that impede interdomain reaction, which is essential in AS-B, and that accommodate beta-lactam formation. The structural data provide the opportunity to alter the synthetic potential of beta-LS, perhaps leading to the creation of new beta-lactamase inhibitors and beta-lactam antibiotics.
Miller
M T
MT
Department of Biochemistry, Northwestern University, Evanston, Illinois 60208, USA.
Bachmann
B O
BO
Townsend
C A
CA
Rosenzweig
A C
AC
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001216:Asparagine / Q000378:metabolism*; D001217:Aspartate-Ammonia Ligase / Q000737:chemistry / Q000145:classification; D001665:Binding Sites; D019818:Clavulanic Acid / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D019143:Evolution, Molecular; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology*; D013329:Structure-Activity Relationship
D000900:Anti-Bacterial Agents; D019818:Clavulanic Acid; D001216:Asparagine; D000255:Adenosine Triphosphate; D000581:Amidohydrolases; C114020:beta-lactam synthetase; D001217:Aspartate-Ammonia Ligase; D008274:Magnesium; C002630:alpha,beta-methyleneadenosine 5'-triphosphate