Mutation of N304 to leucine in Streptomyces clavuligerus deacetoxycephalosporin C synthase creates an enzyme with increased penicillin analogue conversion

Superimposition of deacetoxycephalosporin C synthase (DAOCS) and isopenicil lin N synthase (IPNS) structures revealed that R74, R160, R266 and N304 are strategically located in the catalytic cavity of Streptomyces clavuligerus DAOCS (scDAOCS) and are crucial for orchestrating different substrates. Su bstitutions at these sites to a hydrophobic leucine residue were expected t o stabilize the hydrophobic substrate bound state. Substantial improvements in the biotransformation of penicillin G, ampicillin and amoxicillin to th eir respective cephalosporin moieties were observed using the N304L mutant scDAOCS. Thus, our results have demonstrated the enhancement of scDAOCS act ivity via critical computational analysis and site-directed mutagenesis of endogenous ligands. (C) 2001 Academic Press.