Penicillin expandase

There has also been extensive activity towards the replacement of the entire chemical route to 7-ADCA (Scheme 1.14) with a biocatalytic one. This is somewhat more complex than the above example, as the penicillin fermentation product requires ring expansion as well as side-chain hydrolysis in order to arrive at the desired nucleus. The penicillin nucleus can be converted to the cephalosporin nucleus using expandase enzymes, a process that occurs naturally during the biosynthesis of cephalosporin C by Acremonium chryso-genum and cephamycin C by Streptomyces clavuligems from isopenicUhn N (6-APA containing a 6-L-a-aminoadipoyl side chain). ... 

WK Yeh, JE Dotzlaf, GW Huffman. Biochemical characterization and evolutionary implication of (3-lactam expandase/hydroxylase, expandase and hydroxylase. In H Kleikauf, H vonDohren, eds. 50 Years of Penicillin Application History and Trends. Prague Public, 1994, pp 208-223. 

J Sutherland, R Bovenberg, J van der Laan. Improved process for the production of semi-synthetic cephalosporins via expandase activity on penicillin G. International Patent WO 97/20053, 1997. 

Because of the effectiveness of cephalosporins against penicillin-resistant bacterial pathogens, understanding the substrate specificity, action, and regulation of expandase is of great scientific interest and practical importance. 

The substrate range of the expandase in cell-free extracts using the new conditions included all 15 penicillins tested (Table 2). The largest bioassay zones of inhibition were produced by conversion of penicillins G, X, mX, and 2-thiophenylacetyl-6APA, whereas the smallest zones were obtained with decanoyl-6APA and penicillin V [14],... 

Although DTT is not necessary in the enzymatic conversion of penicillin G [14], this compound is known to stimulate the expandase activity of frozen crude extracts of S. clavuligerus and C. acremonium on penicillin N [40], However, neither this reagent nor P-mercaptoethanol was able to stimulate the subsequent reaction with penicillin G when added during preincubation with FeS04 and ascorbate [43],... 

Homeologous recombination (recombination between partially homologous sequences) was used to produce novel, functional expandase enzymes in vivo) which are hybrids of the S. clavuligerus and N. lactamdurans proteins. DNA sequencing of hybrids obtained in E. coli showed that recombination had occurred at several locations displaying conserved sequences as short as 2 bp. Hybrid genes obtained in a Streptomyces background produced expandases with altered activity on penicillin G, as confirmed by HPLC analysis. 

The biosynthesis of CPC is well known. The first step of CPC synthesis is the formation of the tripeptide S-(L-a-aminoadipyl)-L-cysteinlyl-D-valine (LLD-ACV) from L-a-aminoadipinic acid (AAA), L-cystein (CYS ) and D-valine (VAL) by the LLD-ACV synthetase (ACVS). LLD-ACV is converted with the enzyme isopenicillin AT-synthetase (cyclase) to isopenicillin N (IPN) and then with isopenicillin N-epimerase to penicillin N (PEN). The 6-ring is formed by deacetoxycephalosporin C-synthase (expandase). Deacetoxycephalosporin C (DAOC) is converted with deacetoxycephalosporin C-hydroxylase to decacetylcephalosporin C (DAC) and the latter with deacetylcephalosporin C-acyltransferase to CPC (Fig. 1). Except the amino acid and ACV, all others were monitored by on-line HPLC. 

The use of an expandase to convert penicillins to cephalosporins (9.36) also replaced a more expensive multistep chemical synthesis.238 Penicillin acylase has also been used to remove phenylacetyl protecting groups from amines in the synthesis of oligonucleotides.239... 

Isopenicillin N is a precursor of penicillin, and synthesized from 5-(L-aminoadipoyl)-L-cysteinyl-D-valine by isopenicillin N synthetase11781. Isopenicillin N is then converted into penicillin N by isopenicillin N epimerase. Penicillin N is ring-expanded to deacetoxycepharosporin C by penicillin N expandase. The latter compound is... 

Biochemical studies showed that the oc-aminoadipoyl analogue derived from (2,3)-P-methylenepenam was not a substrate for expandase activity but rather, it was a potent reversible inhibitor of the ring expansion of OC-aminoadipoyl-penicillin into deacetoxycephalosporanic acid by the expandase enzyme... 

A related enzyme, the expandase enzyme, is deployed by cephalosporin-producing organisms to expand the five-membered penicillin ring to the six-membered cephalosporin ring. 

Penicillin N conversion to deacetoxycephalosporin C (DAOC) by expansion of the live-membered thiazolidine ring to a 6 C-dihydrothiazine-ring (DAOC-expandase). 

Synthesis of the 3-lactam antibiotics, particularly cephalosporin C and penicillin N, by Cephalosporium acremonium is inhibited by glucose, ammonium ions, and inorganic phosphate when one or more of these nutrients is present at high concentrations in the medium [94]. Each of these nutrients inhibited the formation and activity of ring expandase, an enzyme catalyzing the conversion of the five-membered ring intermediate isopenicillin N into the six-membered deacetoxycephalosporin C [94]. 

The ring expansion of penicillin N to desacetoxycephalosporin is interesting as well. In spite of intensive efforts, this biosynthesis is stiU not completely clear. On the basis of substrate studies and kinetic investigations, it is assumed, that the chemistry of isopenicUlin N-synthase and expandase are alike and that the ring expansion also proceeds via a ferryl-oxo-intermediate, [41] and that a radical mechanism is involved as well. [42]... 

---