Stereoselective Reduction of Carbonyl Compounds with Actinomycete: Purification and Characterization of Three α-Keto Ester Reductases fromStreptomyces avermitilis

Abstract

We achieved the purification of three alpha-keto ester reductases (Streptomyces avermitilis keto ester reductase, SAKERs-I, -II, and -III) from Streptomyces avermitilis NBRC14893 whole cells. The molecular masses of the native SAKERs-I, -II, and -III were estimated to be 72, 38, and 36 kDa, respectively, by gel filtration chromatography. The subunit molecular masses of SAKERs-I, -II, and -III were also estimated to be 32, 32, and 34 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. The purified SAKERs-II and -III showed a reducing activity for alpha-keto esters (in particular, for ethyl pyruvate). SAKER-I showed a high reducing activity not only toward the alpha- and beta-keto esters, but also toward alpha-keto acid. The N-terminal region amino acid sequences of SAKERs-I, -II, and -III were identical to that of a putative oxidoreductase, SAV2750, a putative oxidoreductase, SAV1849, and a putative oxidoreductase, SAV4117, respectively, hypothetical proteins coded on the S. avermitilis genome.

Purification and characterization of a novel keto ester reductase from the green alga, Chlorella sorokiniana: comparison of enzymological properties with other microbial keto ester reductases

“…CSKER, SAKER-II, and SAKER-III have a monomeric structure, while the other KERs have a dimeric structure; one KER is tetrameric. All the KERs showed a reducing activity towards ethyl 4-chloro-3-oxobutanoate (Ishihara et al 2000b(Ishihara et al , 2004(Ishihara et al , 2008Yamaguchi et al 2002), although CSKER did not reduce a-keto esters to the corresponding hydroxy esters. Thus, the subunit structure and catalytic function of CSKR are more similar to those of the reductases from eukaryotic microbes than to those of prokaryotic keto ester reductases.…”

“…The similarity of the 30 amino acids sequence of the enzyme with those of other proteins was investigated by a computer search of the protein sequence database; no enzyme having a significant sequence similarity to this of CSKER was found. (Nakamura et al 1994) c COBE Reductase, 4-chloro-3-oxobutanoic acid ethyl ester reductase (Wada et al 1988) d Carbonyl Reductase (Kataoka et al 1992) e PsCR, carbonyl reductase (Qi et al 2009) f C., Chlorella; S., Saccharomyces; Ca., Candida; P., Pichia (Yamaguchi et al 2002) f SCKER, keto ester reductase from Streptomyces coelicolor A3(2) (Ishihara et al 2004) g SAKERs, keto ester reductase from Streptomyces avermitilis (Ishihara et al 2008) CSKER comparison with other keto ester reductases Several ethyl 4-chloro-3-oxobutanoate reducing enzymes have been isolated from eukaryotic microorganisms such as Saccharomyces cerevisiae (baker's yeast) (Nakamura et al 1994), Candida magnoliae (Wada et al 1988), Candida macedoniensis (Kataoka et al 1992), and Pichia stipitis (Qi et al 2009). The properties of these enzymes were compared with those of CSKER as shown in Table 4.…”

“…For example, seven yeast keto ester reductases (YKERs) had been isolated from bakers' yeast (Saccharomyces cerevisiae) and their enzymatic properties, including the specific activity, stereoselectivity and the kinetic parameters, were determined (Nakamura et al 1994). Furthermore, seven keto ester reductases (STKER-I, -II, -III, SCKER, SAKER-I, -II, and -III) from three Streptomyces strains had been purified and characterized (Ishihara et al 2000b(Ishihara et al , 2004(Ishihara et al , 2008Yamaguchi et al 2002). However, little information is available on the mechanism underlying the enzymatic reduction of keto esters by Chlorella strains.…”

Microbial Preparation of Chiral Alcohols: Stereoselective Reduction of Carbonyl Compounds using Two Genera of the Streptosporangiaceae Family - Streptosporangium and Nonomuraea

“…As described above, there have been several studies on the biochemical applications of secondary metabolites from actinomycetes. In addition, it has been found that some strains of the genus Streptomyces in the family Streptomycetaceae are useful biocatalysts for the asymmetric reductions of various carbonyl compounds (Ishihara et al, 2015(Ishihara et al, , 2013(Ishihara et al, , 2008(Ishihara et al, , 2004(Ishihara et al, , 2003(Ishihara et al, , 2000(Ishihara et al, , 1997. While this family has thus been extensively studied for the biocatalytic activities of its members, the potential biocatalytic abilities of actinomycetes belonging to other families have not been investigated.…”

Microbial Production of Chiral Hydroxy Esters and Their Analogs: Biocatalytic Reduction of Carbonyl Compounds by Actinobacteria, <i>Agromyces</i> and <i>Gordonia</i> Strains

“…for the biocatalytic activities of their members. Furthermore, several keto ester reductases have been purified from S. thermocyaneoviolaceus, S. coelicolor A3(2), and S. avermitilis whole cells, and the reaction mechanism of the reduction reaction has been elucidated based on the enzymatic properties of these reductases [16] [17] [18]. However, the potential biocatalytic abilities of actinomycetes belonging to other families have not been investigated.…”