Mechanistic studies on isopenicillin N synthase

Abstract

Several plasmids containing the isopenicillin N synthase gene were constructed and introduced into different expression systems that yielded large quantities of protein, mostly as inclusion bodies. Procedures to obtain active IPNS from these expression systems were devised which allowed us to produce large amounts of highly pure enzyme. Several peptide analogs to the natural substrate. 5-L-a-aminoadipovl-L-cysteinyl-D-valine, were synthesized and tested as substrates to evaluate the tolerance of the active site of IPNS from Streptomyces jumonjinensis towards different groups. 13C-Labeled substrate and ACG ,a tripeptide analog of the substrate, were also synthesized as probes for in vitro 13C NMR spectroscopy studies of the cyclization reaction. Low temperature experiments using millimolar concentrations of IPNS with the labeled substrate showed the formation of a shunt metabolite, but no intermediate was detected. Incubation studies with labeled ACG showed the formation of a thioenol compound that has been proposed to be formed by the decomposition of the monolactam intermediate. Structure determination studies of IPNS are currently in progress in a collaborative work with Dr. James Remington. X-ray diffraction quality crystals were obtained in Oregon, capable of diffracting to 2.4 A resolution. Several IPNS-crystal derivatives have been produced, but none of them permitted the solution of the phase problem. The IPNS containing selenomethionine has been produced in our group from a methionine auxotroph of E. coli and has been successfully crystallized in Oregon. These crystals presented the same diffraction properties as the ones from the native enzyme and are currently been studied by multiple-wavelength anomalous dispersion (MAD) in an attempt to solve the crystal structure.