2 ISOZYMES OF CLAVAMINATE SYNTHASE CENTRAL TO CLAVULANIC ACID FORMATION - CLONING AND SEQUENCING OF BOTH GENES FROM STREPTOMYCES-CLAVULIGERUS

Clavaminate synthase (CS) is an alpha-ketoglutarate-dependent oxygenase central to the biosynthesis of clavulanic acid, a potent inhibitor of beta-lactamases. CS catalyzes the oxidative cyclization/desaturation of proclavaminic acid to clavaminic acid in a two-step process involving the intermediacy of dihydroclavaminic acid [Salowe, S. P., Krol, W. J., Iwata-Reuyl, D., & Townsend, C. A. (1991) Biochemistry 30, 2281-2292]. During the purification of CS to homogeneity from Streptomyces clavuligerus [Salowe, S. P., Marsh, E. N., & Townsend, C. A. (I 990) Biochemistry 29, 6499-6508], two forms of the enzyme capable of carrying out the complete reaction having very similar molecular weights and kinetic properties were isolated by Mono-Q chromatography. The gene for each has been cloned, sequenced, and found to be significantly homologous (87% identity). The two genes so isolated, cs1 and cs2, have open reading frames of 975 and 978 nucleotides, respectively, encoding proteins of M(r) 35 347 and 35 774. These genes are located in different loci of the genome separated by > 20 kbp. This separation is large for a natural product biosynthetic pathway in bacteria where gene duplication and limited divergence are typically observed to occur within narrower confines of a gene cluster. Sequence comparisons made between cs1/cs2 and other genes encoding iron-dependent proteins involved in penicillin and cephalosporin biosynthesis in the same organism show minimal homology. Further sequence alignments made to other non-heme iron oxygenases reveal unexpected dissimilarity within the alpha-ketoglutarate-dependent class itself. The limited data available suggests evolutionary convergence among these proteins.