Photoaffinity labelling of isopenicillin N synthetase by laser-flash photolysis

Abstract

Isopenicillin N synthetase (IPNS) from Acremonium chrysogenum was photolabelled by laser-flash photolysis in the presence of a diazirinyl-containing substrate, 2-[3-(3-trifluoromethyl-3H-diazirin-3-yl)-phenoxy]acetyl-S- methyloxycarbonylsulphenyl-L-cysteinyl-D-valine (DCV). Labelling of IPNS by DCV is partially inhibited in the presence of an excess of L-alpha-aminoadipoyl-L-cysteinyl-D-valine (ACV), the natural substrate. In the absence of light, DCV is converted into the corresponding penicillin with comparable Km but significantly depressed Vmax relative to ACV. Selective incorporation of [14C]DCV into IPNS has been demonstrated by fluorography of IPNS analysed by SDS/polyacrylamide-gel electrophoresis. Scintillation counting of labelled IPNS purified on an ion-exchange f.p.l.c. column confirms this result. This methodology may be applicable for studies aimed at investigating the binding of substrates to IPNS.

Histidine-272 of isopenicillin N synthase of Cephalosporium acremonium, which is possibly involved in iron binding, is essential for its catalytic activity

“…Thereafter, the relative influence of substrate binding, cofactor release and product release can be investigated to help in solving the mechanistic function of the enzyme. The milieu towards this target has been concentrated on studying the primary structure of IPNS [8][9][10][11][12][13][14][15], its protein interaction with other molecules to determine the number of binding ligands involved [4][5][6][7], the functional characteristics of ligand binding sites through affinity labelling [24], chemical modification of reactive groups [3], and finally, specific modification of protein structure through site-directed mutagenesis [27]. So far, only site-directed mutagenesis of cysteine residues in clPNS [27] has been carried out in view of their conserved presence in IPNS obtained from various sources.…”

“…The activity of IPNS in cell-free extracts was determined by bioassay using Micrococcus luteus ATCC 318 as the test organism and ACV (a generous gift from Professor J.E. Baldwin of Oxford University, UK) as the substrate [24]. One unit of activity is the amount of enzyme required to form the equivalent of 1 /xmol of isopenicillin N in 1 min.…”

Chiral Bicyclic Tetramates as Non‐Planar Templates for Chemical Library Synthesis

“… Bioassay of products: Microbiological assays were performed by the hole‐plate method with the test organism Staphylococcus aureus N.C.T.C. 6571 or E. coli X580.…”

Post‐Polymerization Modification of Materials using Diaryldiazomethanes: Changes to Surface Macroscopic Properties

“…Although an attempt to increase the peroxide loading density by coupling UHMWPE 4c with diazonium salt 7b gave material 8 , when converted into adduct 9 it did not exhibit any higher loading than the base material. That these loading levels had potential application was suggested by incubation of modified XAD 6a , UHMWPE 6c , and PP 6f with Streptococcus aureus overnight, and these demonstrated substantial levels of antibacterial activity, with kill zones of 38, 37, and 22 mm (a zone of 11 mm is considered to be inactive) (Figure ). That this bioactivity resulted from the combination of surface modification and the presence of hydrogen peroxide was established from appropriate controls; thus, modified polymers 4a , c and f which were not treated with hydrogen peroxide, and unmodified polystyrene XAD‐4, UHMWPE, and PP which were all treated with hydrogen peroxide followed by washing, displayed no biocidal activity in identical assays.…”