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 isopenicillin 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. Substitutions at these sites to a hydrophobic leucine residue were expected to stabilize the hydrophobic substrate bound state. Substantial improvements in the biotransformation of penicillin G, ampicillin and amoxicillin to their respective cephalosporin moieties were observed using the N304L mutant scDAOCS. Thus, our results have demonstrated the enhancement of scDAOCS activity via critical computational analysis and site-directed mutagenesis of endogenous ligands.
Chin
H S
HS
Department of Microbiology, Faculty of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Singapore.
Sim
J
J
Sim
T S
TS
D019943:Amino Acid Substitution; D001216:Asparagine / Q000235:genetics; D001681:Biological Assay; D002851:Chromatography, High Pressure Liquid; D019295:Computational Biology; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007930:Leucine / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013302:Streptomyces / Q000201:enzymology*
D046915:Penicillin-Binding Proteins; D010406:Penicillins; D001216:Asparagine; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase; D007930:Leucine