Biosynthesis of Clavulanic Acid

In the first studies on clavulanic acid biosynthesis Elson and Oliver (279) fed [l- C]-acetate, [2- C]-acetate or [l,2- C]-acetate to cultures of Streptomyces clavuligerus during the clavulanic acid production phase. The resulting samples of C-labelled clavulanic acid were isolated as the benzyl ester (287) and examined by C-n.m.r. spectroscopy. The labelling patterns 

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Li, R., Khaleeli, N. and Townsend, C.A. (2000) 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. The Journal of Bacteriology, 182, 4087—4095. 

Scheme 7.22 Non-heme iron oxygenase in biosynthesis of clavulanic acid.

The common motif shared by non-heme iron oxygenases contains an active site, where two histidines and one carboxylate occupy one face of the Fe(ll) coordination sphere. These enzymes catalyze a variety of oxidative modification of natural products. For example, in the biosynthesis of clavulanic acid, clavaminic acid synthase demonstrates remarkable versatility by catalyzing hydroxylation, oxidative ring formation and desaturation in the presence of a-ketoglutarate (eq. 1 in Scheme 7.22) [80]. The same theme was seen in the biosynthesis of isopenicillin, the key precursor to penicillin G and cephalosporin, from a linear tripeptide proceeded from a NRPS, where non-heme iron oxygenases catalyze radical cyclization and ring expansion (eq. 2 in Scheme 7.22) [81, 82]. 

Baggaley KH, Brown A and Schofield CJ (1997) Chemistry and biosynthesis of clavulanic acid and other clavams. Nat Prod Rep 14, 309-333. 

Structural studies of clavulanic acid dehydrogenase, which catalyzes the biosynthesis of clavulanic acid 54 (R1 = Rz = H) from clavulanate-9-aldehyde, have provided a deeper understanding of the mechanism of the reduction process <2007B1523>. 

The monocyclic lactams include the nocardicins formed by actinomycetes and monobactams formed mostly by bacteria and possessing antibiotic activities with differing sensitivities to /8-lactamases. For biosynthesis of clavulanic acid see Lit. 

Baldwin JE, Adlington RM, Bryans JS, Bringhen O, Coates JB, Crouch NP. Lloyd MD, Schofield CJi Elson SW, Baggaley KH, Casscis R, Nicholson N. Isolation of dihydro-davaminic acid, an intermediate in the biosynthesis of clavulanic acid. Tetrahedron 1991 47 4089-4100. 

Stirling, L, and S. W. Elson Studies on the Biosynthesis of Clavulanic Acid II. Chemical Degradation of C-Labelled Clavulanic Acid. J. Antibiotics 32, 1125 (1979). 

Scheme 10.30 Part of clavulanic acid biosynthesis. Bonds installed byclavaminic acid synthase (CAS) are circled. CAS clavaminic acid synthase. PAH proclavaminic acid amidino hydrolase.

Tahlan, K., Park, H.U., Wong, A., Beatty, P.H. and Jensen, S.E. (2004) Two sets of paralogous genes encode the enzymes involved in the early stages of clavulanic acid and clavam metabolite biosynthesis in streptomyces clavuligenis. 48, 930-939. 

Bushell, M.E., Kirk, S., Zhao, H.-J. and Avignone-Rossa, C.A. (2006) Manipulation of the physiology of clavulanic acid biosynthesis with the aid of metabolic flux analysis. Enzyme and Microbial Technology, 39, 149-157. 

Jensen, S.E., Elder, K.J., Aidoo, K.A. and Paradkar, A.S., (2000) Enzymes catalyzing the early steps of clavulanic acid biosynthesis are encoded by two sets of paralogous genes in Streptomyces clavuligerus. Antimicrobial Agents and Chemotherapy, 44, 720-726. 

P-Lactams. AH 3-lactams are chemically characterized by having a 3-lactam ring. Substmcture groups are the penicillins, cephalosporias, carbapenems, monobactams, nocardicias, and clavulanic acid. Commercially this family is the most important group of antibiotics used to control bacterial infections. The 3-lactams act by inhibition of bacterial cell wall biosynthesis. 

Clavulanic acid has only weak antibacterial activity, but is a potent irreversible inhibitor for many clinically important P-lactamases (10—14,57,58) including penases, and Richmond-Sykes types 11, 111, IV, V, VI ([Bacteroides). Type I Cephases are poorly inhibited. Clavulanic acid synergizes the activity of many penicillins and cephalosporins against resistant strains. The chemistry (59—63), microbiology (64,65), stmcture activity relationships (10,13,60—62,66), biosynthesis (67—69), and mechanism of action (6,26,27,67) have been reviewed. 

Garbapenem P-Lactamase Inhibitors. Carbapenems are another class of natural product P-lactamase inhibitors discovered about the same time as clavulanic acid. Over forty naturally occurring carbapenems have been identified many are potent P-lactamase inhibitors. Garbapenem is the trivial name for the l-a2abicyclo[3.2.0]hept-2-ene ring system (21) shown in Table 3. The synthesis (74), biosynthesis (75), and P-lactamase inhibitory properties (13,14,66) of carbapenems have been reviewed. Carbapenems are often more potent than clavulanic acid and include type I Cephases in the spectmm of inhibition. Table 3 Hsts the available P-lactamase inhibition data. Synergy is frequendy difficult to demonstrate because the compounds are often potent antibacterials. 

Other examples of a-keto acid-dependent enzymes are mammalian proline hydroxylase and bacterial clavaminate synthase [113]. The latter enzyme is of particular interest as it is responsible for the catalysis of three individual steps in the biosynthesis of the (3-lactamase inhibitor clavulanic acid (Scheme 10.30). 

Figure 12.2 The biosynthesis of the clavulanic acid pathway [77]. (Reproduced by permission from Macmillan Publishers Ltd M.C.Y. Chang and J.D. Keasling. Production of isoprenoid pharmaceuticals by engineered microbes. Nature Chemical Biology 2 (12) 674-682. London Nature Publishing Group. 2006 Macmillan)...

Aidoo, K.A., Wong, A., Alexander, D.C. et al. (1994) Cloning, sequencing and disruption of a gene from Streptomyces clavuligerus involved in clavulanic acid biosynthesis. Gene, 147, 41—46. 

Scheme 2.2 Examples of reactions catalyzed by and RNA by the protein AlkB [54] (R = sugar al

Butterworth, D. (1984). Clavulanic acid Properties, biosynthesis, and fermentation. In Biotechnology of Industrial Antibiotics (E. J. Vandamme, ed.), pp. 225-235. Dekker, New York. 

Topics covered in this b3ok include the partial and total synthesis, biosynthesis, and structure-activity relationships of the principle classes of /3-lactam antibiotics, including penicillins, cepfialosporins, clavulanic acids and olivanic acids, and their interactions with bacteria. 

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