Clavulanic Acid Dehydrogenase: Structural and Biochemical Analysis of the Final Step in the Biosynthesis of the -Lact
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- 作者：Alasdair K. MacKenzie ; Nadia J. Kershaw ; Helena Hernandez ; Carol V. Robinson ; Christopher J. Schofield ; Inger Andersson
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：February 13, 2007
- 年：2007
- 卷：46
- 期：6
- 页码：1523 - 1533
- 全文大小：704K
- 年卷期：v.46,no.6(February 13, 2007)
- ISSN：1520-4995
文摘
The ultimate step in the biosynthesis of the medicinally important 
-lactamase inhibitorclavulanic acid is catalyzed by clavulanic acid dehydrogenase (CAD). CAD is responsible for the NAPDH-dependent reduction of the unstable intermediate clavulanate-9-aldehyde to yield clavulanic acid. Here,we report biochemical and structural studies on CAD. Biophysical analyses demonstrate that CAD existsas dimeric and tetrameric species in solution. The reaction performed by CAD was shown to be reversible,allowing the use of clavulanic acid for activity analyses. The crystal structure of CAD was solved usingsingle-wavelength anomalous diffraction with a seleno-methionine derivative. The structure reveals thatthe individual monomers comprise a single domain possessing the Rossmann fold, characteristic ofdinucleotide-binding enzymes. The monomers are arranged as tetramers, similar to other tetrameric membersof the short-chain dehydrogenase/reductase family. The structure of the unreactive complex of CAD withclavulanic acid and NADPH suggests how CAD is able to catalyze the reduction of clavulanate-9-aldehydewithout fragmentation of the bicyclic -lactam ring structure. The relative positions of NADPH andclavulanic acid, in the active site, together with the presence of the latter in an eclipsed conformation,rationalizes previous labeling studies demonstrating that the incorporation of the C5 pro-R, but not pro-S,hydrogen of ornithine/arginine into the C9 position of clavulanic acid occurs with overall inversion ofconfiguration.
-lactamase inhibitorclavulanic acid is catalyzed by clavulanic acid dehydrogenase (CAD). CAD is responsible for the NAPDH-dependent reduction of the unstable intermediate clavulanate-9-aldehyde to yield clavulanic acid. Here,we report biochemical and structural studies on CAD. Biophysical analyses demonstrate that CAD existsas dimeric and tetrameric species in solution. The reaction performed by CAD was shown to be reversible,allowing the use of clavulanic acid for activity analyses. The crystal structure of CAD was solved usingsingle-wavelength anomalous diffraction with a seleno-methionine derivative. The structure reveals thatthe individual monomers comprise a single domain possessing the Rossmann fold, characteristic ofdinucleotide-binding enzymes. The monomers are arranged as tetramers, similar to other tetrameric membersof the short-chain dehydrogenase/reductase family. The structure of the unreactive complex of CAD withclavulanic acid and NADPH suggests how CAD is able to catalyze the reduction of clavulanate-9-aldehydewithout fragmentation of the bicyclic -lactam ring structure. The relative positions of NADPH andclavulanic acid, in the active site, together with the presence of the latter in an eclipsed conformation,rationalizes previous labeling studies demonstrating that the incorporation of the C5 pro-R, but not pro-S,hydrogen of ornithine/arginine into the C9 position of clavulanic acid occurs with overall inversion ofconfiguration.