Glutamine-230 influences enzyme solubility but not catalysis in Streptomyces clavuligerus isopenicillin N synthase

The conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isope nicillin N is dependent upon the catalytic action of isopenicillin N syntha se (IPNS), an important enzyme in the penicillin and cephalosporin biosynth etic pathway. Recent catalytic investigations on the conserved glutamine-23 0 in the bacterial Streptomyces jumonjinensis IPNS and the corresponding gl utamine-234 in the fungal Cephalosporium acremonium IPNS showed contrasting results whereby the former was suggested to be essential for IPNS activity whereas the latter was found not to be so. In order to unravel these confl icting results; we report the site-directed mutagenesis investigation on th e corresponding glutamine-230 in a third IPNS isozyme, which is the bacteri al Streptomyces clavuligerus IPNS (scIPNS). IPNS enzymatic assays showed th at catalytic activity of the mutant Q230L scIPNS was reduced but not elimin ated. Moreover, the solubility of the mutant enzyme was also markedly reduc ed. Hence, we can conclude that glutamine-230 in scIPNS is not essential fo r catalysis and correspondingly in all IPNS. (C) 1999 Federation of Europea n Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.