ENZYMATIC-SYNTHESIS OF DIASTEREOSPECIFIC CARBACEPHEM INTERMEDIATES USING SERINE HYDROXYMETHYLTRANSFERASE

The serine hydroxymethyltransferase (SHMT) gene glyA was over-expresse d in Escherichia coli and the enzyme was purified to near homogeneity, Reaction conditions for E. coli and rabbit liver SHMTs were optimized using succinic semialdehyde methyl ester (SSAME) and glycine, The cat alytic efficiency (k(cat)/K-m) of E. coil SHMT for SSAME was 2.8-fold higher than that of rabbit liver enzyme. E. coil SHMT displayed a pH-d ependent product distribution different from that of rabbit liver enzy me, For the pyridoxal-5'-phosphate (PLP)-dependent reaction, E. coil a nd rabbit liver SHMTs showed a high product diastereospecificity. The stoichiometric ratio of PLP to the dimeric E. coli SHMT was 0.5-0.7, i ndicating a requirement for external PLP for maximal activity, Using S SAME or its analog at a high temperature, E. coil SHMT mediated effici ent condensation via a lactone pathway, In contrast, at a low temperat ure, the enzyme catalyzed efficient conversion of 4-penten-1-al via a non-lactone mechanism. Efficient conversion of either aldehyde type to a desirable diastereospecific product was observed at a pilot scale, E. coil SHMT exhibited a broad specificity toward aldehyde substrates; thus it can be broadly useful in chemo-enzymatic synthesis of a chira l intermediate in the manufacture of an important carbacephem antibiot ic.