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
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.