ANALYSIS OF THE CONVERSION OF HA-AMINOADIPOYL)-L-CYSTEINYL-D-ALPHA-AMINOBUTYRATE BY ACTIVE-SITE MUTANTS OF ASPERGILLUS-NIDULANS ISOPENICILLIN-N SYNTHASE

Background: Penicillins and cephalosporins constitute a major class of clinically useful antibiotics. A key step in their biosynthesis invol ves the oxidative cyclisation of delta-(L-alpha-aminoadipoyl)-L-cystei nyl-D-valine to isopenicillin N by isopenicillin N synthase (IPNS). Th is chemically remarkable transformation has been extensively studied u sing substrate analogues, The conversion of an analogue in which the v aline is replaced by a-aminobutyrate results in three products, two ep imeric penams and a cepham. The ratio of these products in reactions c atalysed by four different IPNS isozymes has been used previously to p robe the thermicity of the chemical mechanism. But how IPNS restricts the products from the natural substrate to a single penam (isopenicill in N) has remained unknown. Results: A key active-site residue, Leu223 , identified according to a model of enzyme-substrate binding, has bee n altered to sterically less demanding residues, As the steric constra ints on the upper part of the active site are reduced, the ratio of th e beta-methyl penam to the cepham increases when the alpha-aminobutyra te-containing substrate analogue is used. These results suggest a mech anism for processing of the natural substrate in which IPNS uses steri c control to restrict the conformational freedom of an intermediate su ch that the only product is the penam. Conclusions: Using steric press ure to control conformation, and hence to disfavour reactions leading to alternate products, is probably the result of evolutionary selectio n for a biologically active product at the expense of biologically ina ctive byproducts. It is likely that this sort of enzymatic catalysis i s used in situations where substrate conversion is highly exothermic a nd a variety of products are possible.