title: Structural and mechanistic studies of the orf12 gene product from the clavulanic acid biosynthesis pathway.
abstract: Structural and biochemical studies of the orf12 gene product (ORF12) from the clavulanic acid (CA) biosynthesis gene cluster are described. Sequence and crystallographic analyses reveal two domains: a C-terminal penicillin-binding protein (PBP)/β-lactamase-type fold with highest structural similarity to the class A β-lactamases fused to an N-terminal domain with a fold similar to steroid isomerases and polyketide cyclases. The C-terminal domain of ORF12 did not show β-lactamase or PBP activity for the substrates tested, but did show low-level esterase activity towards 3'-O-acetyl cephalosporins and a thioester substrate. Mutagenesis studies imply that Ser173, which is present in a conserved SXXK motif, acts as a nucleophile in catalysis, consistent with studies of related esterases, β-lactamases and D-Ala carboxypeptidases. Structures of wild-type ORF12 and of catalytic residue variants were obtained in complex with and in the absence of clavulanic acid. The role of ORF12 in clavulanic acid biosynthesis is unknown, but it may be involved in the epimerization of (3S,5S)-clavaminic acid to (3R,5R)-clavulanic acid.
authors.affiliation: Department of Molecular Biology, Swedish University of Agricultural Sciences, Box 590, S-751 24 Uppsala, Sweden.
authors.forename: Karin
authors.initials: K
authors.lastname: Valegård
authors.forename: Aman
authors.initials: A
authors.lastname: Iqbal
authors.forename: Nadia J
authors.initials: NJ
authors.lastname: Kershaw
authors.forename: David
authors.initials: D
authors.lastname: Ivison
authors.forename: Catherine
authors.initials: C
authors.lastname: Généreux
authors.forename: Alain
authors.initials: A
authors.lastname: Dubus
authors.forename: Cecilia
authors.initials: C
authors.lastname: Blikstad
authors.forename: Marina
authors.initials: M
authors.lastname: Demetriades
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Hopkinson
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Lloyd
authors.forename: David I
authors.initials: DI
authors.lastname: Roper
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
authors.forename: Inger
authors.initials: I
authors.lastname: Andersson
authors.forename: Michael A
authors.initials: MA
authors.lastname: McDonough
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002268:Carboxypeptidases / Q000378:metabolism; D020134:Catalytic Domain; D002511:Cephalosporins / Q000378:metabolism; D019818:Clavulanic Acid / Q000096:biosynthesis* / Q000737:chemistry; D018360:Crystallography, X-Ray; D006868:Hydrolysis; D008958:Models, Molecular; D010406:Penicillins / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012694:Serine / Q000235:genetics; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism; D047090:beta-Lactams / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002511:Cephalosporins; D010406:Penicillins; D047090:beta-Lactams; D019818:Clavulanic Acid; D012694:Serine; D002268:Carboxypeptidases; D001618:beta-Lactamases
keywords: clavulanic acid biosynthesis pathway; esterases; penicillin-binding proteins; β-lactam antibiotics; β-lactamase fold