Skip to main content
Research article - Peer-reviewed, 2013

Structural and mechanistic studies of the orf12 gene product from the clavulanic acid biosynthesis pathway

Valegård, Karin; Iqbal, Aman; Kershaw, Nadia J.; Ivison, David; Genereux, Catherine; Dubas, Alain; Blikstad, Cecilia; Demetriades, Marina; Hopkinson, Richard J.; Lloyd, Adrian J.; Roper, David I.; Schofield, Christopher J.; Andersson, Inger; McDonough, Michael A.


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)/beta-lactamase-type fold with highest structural similarity to the class A beta-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 beta-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, beta-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.

Published in

Acta Crystallographica Section D: Biological Crystallography
2013, volume: 69, pages: 1567-1579

Authors' information

Valegård, Karin
Swedish University of Agricultural Sciences, Department of Molecular Biology
Iqbal, Aman
University of Oxford
Kershaw, Nadia J.
University of Oxford
Ivison, David
University of Oxford
Genereux, Catherine
University of Liège
Dubas, Alain
University of Liège
Blikstad, Cecilia
Uppsala University
Demetriades, Marina
University of Oxford
Hopkinson, Richard J.
University of Oxford
Lloyd, Adrian J.
University of Warwick
Roper, David I.
University of Warwick
Schofield, Christopher J.
University of Oxford
Andersson, Inger
Uppsala University
McDonough, Michael A.
University of Oxford

UKÄ Subject classification

Biochemistry and Molecular Biology

Publication Identifiers


URI (permanent link to this page)