Open AccessMutational evidence for the role of serine‐283 in Cephalosporium acremonium isopenicillin N synthase
Author(s) -
Loke Paxton,
Sim TiowSuan
Publication year - 1998
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1998.tb13169.x
Subject(s) - acremonium , aspergillus nidulans , tripeptide , biochemistry , serine , active site , stereochemistry , biology , cephalosporin c , amino acid , site directed mutagenesis , mutant , mutagenesis , arginine , enzyme , chemistry , cephalosporin , botany , gene , antibiotics
Creation of isopenicillin N from δ‐( l ‐α‐aminoadipyl)‐ l ‐cysteinyl‐ d ‐valine (ACV) in the penicillin and cephalosporin biosynthetic pathway is catalysed by isopenicillin N synthase (IPNS), a non‐heme iron‐containing dioxygenase. A tripeptide R‐X‐S motif which consists of arginine‐281 and serine‐283 ( Cephalosporium acremonium IPNS numbering) was found to be conserved in IPNS and other related proteins. These two amino acids mentioned were proposed to have a role in ACV substrate binding by the recent Aspergillus nidulans IPNS crystal structure. Using site‐directed mutagenesis, arginine‐281 in C. acremonium IPNS (cIPNS) was earlier found to be essential for catalysis by our group. Similarly, serine‐283 in cIPNS was also altered by site‐directed mutagenesis to determine its role in cIPNS. No measurable activity was detected from the resultant mutant using enzyme bioassays. It is most likely that the elimination of the mutant's substrate‐binding capability similar to that of arginine‐281 lead to the abolishment of the catalytic reaction. This highlights the importance of the R‐X‐S motif in the functionality of cIPNS.