Chapter 16. Enzymology of beta-lactam compounds with cephem structure produced by actinomycete.

Cephamycins are beta-lactam antibiotics with a cephem structure produced by actinomycetes. They are synthesized by a pathway similar to that of cephalosporin C in filamentous fungi but the actinomycetes pathway contains additional enzymes for the formation of the alpha-aminoadipic acid (AAA) precursor and for the final steps specific to cephemycins. Most of the biochemical and genetic studies on cephemycins have been made on cephemycin C biosynthesis in the producer strains Streptomyces clavuligerus ATCC27064 and Amycolatopsis lactamdurans NRRL3802. Genes encoding cephamycin C biosynthetic enzymes are clustered in both actinomycetes. Ten enzymatic steps are involved in the formation of cephamycin C. The precursor alpha-AAA is formed by the sequential action of lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase. Steps common to cephalosporin C biosynthesis include the formation of the tripeptide L-delta-alpha-aminoadipyl-L-cysteinyl-D-valine (ACV) by ACV synthetase, the cyclization of ACV to form isopenicillin N (IPN) by IPN synthase, the epimerization of IPN to penicillin N by isopenicillin N epimerase, the ring expansion of penicillin N to a six member cephem ring by deacetoxycephalosporin C synthase (DAOCS) and the hydroxylation at C-3' by deacetylcephalosporin C hydroxylase. However, in actinomycetes, the epimerization step is different from that in cephalosporin-producing fungi, and the expansion of the ring and its hydroxylation are performed by separate enzymes. Specific steps in cephamycin biosynthesis include the carbamoylation at C-3' by cephem carbamoyl transferase and the introduction of a methoxyl group at C-7 by the joint action of a C-7 cephem-hydroxylase and a methyltransferase. All the enzymes of the pathway have been purified almost to homogeneity and the DAOC synthase and 7-hydroxycephem-methyltransferase (CmcI) of S. clavuligerus have been crystallized giving insights into the mode of action of these enzymes. The cefE gene of S. clavuligerus, encoding DAOCS, has been extensively used to expand the penicillin ring in filamentous fungi in vivo using DNA recombinant technology.