UNDERSTANDING THE SUBSTRATE BINDING MECHANISM OF STREPTOMYCES CLAVULIGERUS DEACETOXYCEPHALOSPORIN C SYNTHASE FOR BIOSYNTHESIS OF NOVEL CEPHALOSPORINS

GOO KIAN SIM

Publication

UNDERSTANDING THE SUBSTRATE BINDING MECHANISM OF STREPTOMYCES CLAVULIGERUS DEACETOXYCEPHALOSPORIN C SYNTHASE FOR BIOSYNTHESIS OF NOVEL CEPHALOSPORINS

GOO KIAN SIM

Abstract

Streptomyces clavuligerus deacetoxycephalosporin C synthase (scDAOCS) is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid, a precursor for the production of semi-synthetic cephalosporins. However, the substrate affinity of scDAOCS, in the conversion of penicillins to corresponding expanded cephalosporins, has limitations. Thus, the main objective in this study was to manipulate scDAOCS via understanding the molecular basis of its functionality and to forge it to accommodate a wider spectrum of substrates for enhanced conversion into new antimicrobial products. To this end, a cohort of site-directed mutated scDAOCS proteins were made and investigated for changes to their prowess in transforming penicillins, including ampicillin, carbenicillin and phenethicillin, to cephalosporin moieties. Results of the study on the involvement of amino acid residues, Y184, L186, V275, C281, W297, I298, V303 and the C-terminal residues between N304-R307, were found to be most prominent in affecting the entry of selected substrates, enhancing substrate binding affinities and improving catalytic activities up to 283% of the wild-type enzyme activity . As such, the possibility of transforming existing penicillins into new cephalosporins, as potential antibacterials, looks promising. To understand the effects of combining various beneficial mutations, selected mutations were also paired and analysed. The bioassay results showed that the C-terminal mutations, i.e., N304A, N304L, N304M, N304K, N304R, I305M, R306L and R307L, in combination with C281Y or V275I substantially increased the catalytic activities further by up to 1,347% of the wild-type enzyme activity. Based on these studies, it was clear that double mutants had enhanced substrate binding affinities compared to single mutants. The understanding of the substrate binding mechanism of scDAOCS was also exploited for the investigation of the substrate selectivity of another biosynthetic enzyme, deacetylcephalosporin C synthase (DACS), which catalyses a hydroxylation reaction. Genetic modifications of its C-terminal residues T308 and H310 conferred a ring-expansion activity to provide this additional function to DACS. This finding suggested that the C-terminal of DACS is likely to be involved in substrate selectivity leading to the type of enzymatic activity it catalyses. This study supports the implication of mobilizing the substrate binding properties of biosynthetic enzymes to tailor desired bioactive products.