| The long sought primary metabolic C3 precursor to the β-lactam carbons of clavulanic acid has been identified as D-glyceraldehyde-3-phosphate. In a novel N-C bond forming thiamine pyrophosphate-mediated reaction catalyzed by ORF2, encoded by the first gene of the clavulanic acid biosynthetic cluster, this glycolytic intermediate is coupled to L-arginine. The product of this unusual reaction is the first dedicated intermediate in clavulanic acid biosynthesis - N2-(carboxyethyl)-L-arginine.; The facial 2-His-1 carboxylate (Asp/Glu) motif has emerged as the structural paradigm for metal binding in the (α-ketoglutarate-dependent nonheme iron oxygenases. The ferrous active site of ciavaminate synthase, an unusual member of this enzyme family that mediates three different, nonsequential reactions during the biosynthesis of clavulanic acid, was examined by site-directed mutagenesis. Two ligands to iron, histidine145 and histidine 280, were identified. Neighboring histidine 145, a third ligand, glutamate 147, was tentatively identified based on sequence homologies to a known His-1 motif and the His-Xaa-Asp/Glu signature. The separation of His145 and His280 is more than twice that seen in the current 2-His-1-carboxylate model and sequence analysis reveals a subclass of non-heme iron oxygenases with similar ferrous active site construction. In this Work, an alternative iron binding motif was recognized, which we propose as His-3.; Previously it was thought that eight contiguous genes within the genome of Streptomyces clavuligerus were sufficient for clavulanic acid biosynthesis because they allowed production of the antibiotic in a heterologous host. In contrast, three new genes—orf10(cyp ), orf11(fd) and orf12 —that are required for clavulanic acid biosynthesis as indicated by gene replacement and transcomplementation have been identified. These genes are located immediately downstream of orf9(cad) in the clavulanic acid biosynthetic gene cluster. These results extend the clavulanic acid gene cluster and attest to the importance of analyzing biosynthetic genes in the context of their natural host. The potential role in the pathway has been proposed for these genes based on sequence analysis. |
