Expandase

Biochemical studies showed that the a-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 (7.-aminoadipoy1-penici11in into deacetoxycephalosporanic acid by the expandase enzyme... 

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). ... 

S Kovacevic, JR Miller. Cloning and sequencing of the (3-lactam hydroxylase gene (ce/F) from Streptomyces clavuligerus gene duplication may have led to separate hydroxylase and expandase activities in the actinomycetes. J Bacteriol 173 398-400, 1991. 

SM Samson, JE Dotzlaf, ML Slisz, GW Becker, RM Van Frank, LE Veal, WK Yeh, JR Miller, SW Queener, TD Ingolia. Cloning and expression of the fungal expandase/hydroxylase gene involved in cephalosporin biosynthesis. Bio/Technol-ogy 5 1207-1214, 1987. 

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. 

MJ Conder, PC McAda, JA Rambosek. Recombinant expandase bioprocess for preparing 7-aminodesacetoxycephalosporanic acid (7-ADCA). U.S. Patent 5,318,896, 1994. 

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 crystal structure of the S. clavuligerus expandase has been recently reported [31] and, based on it, a mechanism for the formation of the reactive... 

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],... 

Table 2 Substrate Specificity of Expandase Using Cell-Free Extracts...

Cell-free extracts were preincubated in buffer in the cold or at 30°C for 2 hr to assess the stability of the expandase over time [43]. After 2 hr, the remaining components of the reaction mixture were added and the tubes were incubated under standard conditions for another 3 hr. In all cases, the initial rates were similar to that of the control (no preincubation) and amounts of product obtained were almost equal to the control value. Thus the lack of activity after 2-3 hr could not be attributed to enzyme instability during shaking at 30°C. When Fe3+ (as ferric sulfate) was substituted for Fe2+ in the reaction, activity was unaffected. Therefore, inactivation was apparently not due to oxidation of Fe2+. 

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],... 

IV. IN VIVO CONSTRUCTION OF HYBRID EXPANDASES BY HOMEOLOGOUS RECOMBINATION... 

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