Enzymatic Properties of KER

Asako et al. found a novel reductase in P. citrinum IF04631 18,52], which could reduce methyl 4-bromo-3-oxobutyrate (BAM) to methyl (S)-4-bromo-3-hydroxybu-tyrate (BHBM), a usefiil pharmaceutical intermediate for the synthesis of HMG-CoA reductase inhibitors as wdl as (S)-4 hlroro-3-hydroxybutyrate (CHBE), and doned the enzyme s gene. The reactivity of BHBM as a chiral intermediate is much higher than CHBE because of the existence of bromine atom however, BHBM is quite toxic for biocatalysts. Therefore, the enzyme catalyzing the reduction of BAM should possess tolerance toward such compounds. 

KER is a monomeric enzyme whose molecular weight is 36.7 kDa (325 amino acids) and theoretical isoelectric point (pi) is 6.21 (Table 6.3). Purified recombinant KER catalyzes the reduction of BAM and ethyl 4-chloroacetoacetate (CAE) into BHBM and CHBE, respectively, in the presence of NADPH, while NADH does not 

coli cells containing recombinant KER converts BAM into the (S)-form of methyl 4-bromo-3-hydroxybutanoate (BHBM) with 97.1% ee, and CAE into methyl (S)-4-chloro-3-hydroxybutanoate (CHBE) with 63.1% ee, indicating a little dissatisfied result for ee of produced chiral alcohols. 

The optimum pH ofthe reaction ofpurified KER is pH 6.0-6.5, and the pH stability oftheenzymeisinapH range from6to 10. The thermal stability ofKERexamined by incubation for 20 min in the 0.1 M buffer (pH 7.0) is as follows 75% activity at 40 °C and 0% at above 50 °C. Thus, the enzyme was not so thermally stable. 

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