FUNCTIONAL-ANALYSIS OF A CONSERVED ASPARTATE D218 IN CEPHALOSPORIUM-ACREMONIUM ISOPENICILLIN N SYNTHASE

Authors
Citation
P. Loke et al., FUNCTIONAL-ANALYSIS OF A CONSERVED ASPARTATE D218 IN CEPHALOSPORIUM-ACREMONIUM ISOPENICILLIN N SYNTHASE, FEMS microbiology letters, 157(1), 1997, pp. 137-140
Citations number
15
Journal title
ISSN journal
03781097
Volume
157
Issue
1
Year of publication
1997
Pages
137 - 140
Database
ISI
SICI code
0378-1097(1997)157:1<137:FOACAD>2.0.ZU;2-V
Abstract
Isopenicillin N synthase (IPNS) is instrumental in the catalytic conve rsion of a tripeptide precursor delta-(L-alpha-aminoadipyl)-L-cysteiny l-D-valine to a bioactive intermediate isopenicillin N in the beta-lac tam antibiotic biosynthetic pathway. It has recently been shown that t his reaction is dependent on a conserved aspartate, D214, in a bacteri al Streptomyces jumonjinensis IPNS. Thus, this study was carried out t o provide the experimental evidence for the involvement of a similarly conserved aspartate residue, D218, in a fungal Cephalosporium acremon ium IPNS (cIPNS). Initially, alteration of the aspartate residue to ge nerate the mutant D218L cIPNS protein was achieved by site-directed mu tagenesis. Subsequent enzyme assays indicated that the catalytic prope rty of the mutant protein was lost, attesting to the need for the corr esponding conserved aspartate to maintain IPNS functionality. It is al so evident from the observed results that site-directed mutagenesis of this particular aspartate residue in cIPNS can affect its solubility. It is therefore important to take these potential changes into consid eration when site-directed mutant proteins are analysed for catalytic function.