Dissociation of cephamycin C and clavulanic acid biosynthesis by 1,3-diaminopropane inStreptomyces clavuligerus

ABSTRACT An approximately 12.5-kbp region of DNA sequence from beyond the end of the previously described clavulanic acid gene cluster was analyzed and found to encode nine possible open reading frames (ORFs). Involvement of these ORFs in clavulanic acid biosynthesis was assessed by creating mutants with defects in each of the ORFs. orf12 and orf14 had been previously reported to be involved in clavulanic acid biosynthesis. Now five additional ORFs are shown to play a role, since their mutation results in a significant decrease or total absence of clavulanic acid production. Most of these newly described ORFs encode proteins with little similarity to others in the databases, and so their roles in clavulanic acid biosynthesis are unclear. Mutation of two of the ORFs, orf15 and orf16, results in the accumulation of a new metabolite, N-acetylglycylclavaminic acid, in place of clavulanic acid. orf18 and orf19 encode apparent penicillin binding proteins, and while mutations in these genes have minimal effects on clavulanic acid production, their normal roles as cell wall biosynthetic enzymes and as targets for β-lactam antibiotics, together with their clustered location, suggest that they are part of the clavulanic acid gene cluster.

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Expansion of the Clavulanic Acid Gene Cluster: Identification and In Vivo Functional Analysis of Three New Genes Required for Biosynthesis of Clavulanic Acid by Streptomyces clavuligerus

Journal of Bacteriology ◽

10.1128/jb.182.14.4087-4095.2000 ◽

2000 ◽

Vol 182 (14) ◽

pp. 4087-4095 ◽

Cited By ~ 40

Author(s):

Rongfeng Li ◽

Nusrat Khaleeli ◽

Craig A. Townsend

Keyword(s):

Clavulanic Acid ◽

Gene Cluster ◽

Streptomyces Clavuligerus ◽

Biosynthetic Gene Cluster ◽

Gene Replacement ◽

Resistant Bacteria ◽

Acid Biosynthesis ◽

Significant Similarity ◽

New Genes

ABSTRACT Clavulanic acid is a potent inhibitor of β-lactamase enzymes and is of demonstrated value in the treatment of infections by β-lactam-resistant bacteria. Previously, it was thought that eight contiguous genes within the genome of the producing strainStreptomyces clavuligerus were sufficient for clavulanic acid biosynthesis, because they allowed production of the antibiotic in a heterologous host (K. A. Aidoo, A. S. Paradkar, D. C. Alexander, and S. E. Jensen, p. 219–236, In V. P. Gullo et al., ed., Development in industrial microbiology series, 1993). In contrast, we report the identification of three new genes, orf10 (cyp), orf11(fd), and orf12, that are required for clavulanic acid biosynthesis as indicated by gene replacement andtrans-complementation analysis in S. clavuligerus. These genes are contained within a 3.4-kb DNA fragment located directly downstream of orf9(cad) in the clavulanic acid cluster. While theorf10 (cyp) and orf11(fd) proteins show homologies to other knownCYP-150 cytochrome P-450 and [3Fe-4S] ferredoxin enzymes and may be responsible for an oxidative reaction late in the pathway, the protein encoded by orf12 shows no significant similarity to any known protein. The results of this study extend the biosynthetic gene cluster for clavulanic acid and attest to the importance of analyzing biosynthetic genes in the context of their natural host. Potential functional roles for these proteins are proposed.

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A pathway-specific transcriptional activator regulates late steps of clavulanic acid biosynthesis inStreptomyces clavuligerus

Molecular Microbiology ◽

10.1046/j.1365-2958.1998.00731.x ◽

1998 ◽

Vol 27 (4) ◽

pp. 831-843 ◽

Cited By ~ 57

Author(s):

Ashish S. Paradkar ◽

Kwamena A. Aidoo ◽

Susan E. Jensen

Keyword(s):

Clavulanic Acid ◽

Transcriptional Activator ◽

Acid Biosynthesis

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Streptomyces clavuligerus relA-null mutants overproduce clavulanic acid and cephamycin C: negative regulation of secondary metabolism by (p)ppGpp

Microbiology ◽

10.1099/mic.0.2007/011890-0 ◽

2008 ◽

Vol 154 (3) ◽

pp. 744-755 ◽

Cited By ~ 36

Author(s):

Juan P. Gomez-Escribano ◽

Juan F. Martín ◽

A. Hesketh ◽

M. J. Bibb ◽

P. Liras

Keyword(s):

Clavulanic Acid ◽

Secondary Metabolism ◽

Streptomyces Clavuligerus ◽

Negative Regulation ◽

Cephamycin C ◽

Null Mutants

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X-ray crystal structure of ornithine acetyltransferase from the clavulanic acid biosynthesis gene cluster

Biochemical Journal ◽

10.1042/bj20040814 ◽

2005 ◽

Vol 385 (2) ◽

pp. 565-573 ◽

Cited By ~ 22

Author(s):

Jonathan M. ELKINS ◽

Nadia J. KERSHAW ◽

Christopher J. SCHOFIELD

Keyword(s):

Crystal Structure ◽

Clavulanic Acid ◽

Gene Cluster ◽

Acid Biosynthesis ◽

Biosynthesis Gene Cluster ◽

Reading Frame ◽

X Ray ◽

Orf6 Gene ◽

Biosynthesis Gene ◽

Acetyl Transfer

The orf6 gene from the clavulanic acid biosynthesis gene cluster encodes an OAT (ornithine acetyltransferase). Similar to other OATs the enzyme has been shown to catalyse the reversible transfer of an acetyl group from N-acetylornithine to glutamate. OATs are Ntn (N-terminal nucleophile) enzymes, but are distinct from the better-characterized Ntn hydrolase enzymes as they catalyse acetyl transfer rather than a hydrolysis reaction. In the present study, we describe the X-ray crystal structure of the OAT, corresponding to the orf6 gene product, to 2.8 Å (1 Å=0.1 nm) resolution. The larger domain of the structure consists of an αββα sandwich as in the structures of Ntn hydrolase enzymes. However, differences in the connectivity reveal that OATs belong to a structural family different from that of other structurally characterized Ntn enzymes, with one exception: unexpectedly, the αββα sandwich of ORF6 (where ORF stands for open reading frame) displays the same fold as an DmpA (L-aminopeptidase D-ala-esterase/amidase from Ochrobactrum anthropi), and so the OATs and DmpA form a new structural subfamily of Ntn enzymes. The structure reveals an α2β2-heterotetrameric oligomerization state in which the intermolecular interface partly defines the active site. Models of the enzyme–substrate complexes suggest a probable oxyanion stabilization mechanism as well as providing insight into how the enzyme binds its two differently charged substrates.

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