Altering the Substrate Specificity of Reductase CgKR1 from Candida glabrata by Protein Engineering for Bioreduction of Aromatic α‐Keto Esters

Abstract

A versatile keto ester reductase CgKR1, exhibiting a broad substrate spectrum, was obtained from Candida glabrata by genome data mining. It showed the highest activity toward an aliphatic bketo ester, ethyl 4-chloro-3-oxobutanoate (COBE), but much lower activity toward bulkier a-keto esters with an aromatic group, such as methyl ortho-chlorobenzoylformate (CBFM) and ethyl 2oxo-4-phenylbutyrate (OPBE). By rational design of the active pocket, the substrate specificity of the reductase was significantly altered and this tailormade reductase showed a much higher activity toward aromatic a-keto esters (~7-fold increase in k cat /K m toward CBFM) and lower activity toward aliphatic keto esters (~12-fold decrease in k cat /K m toward COBE). Meanwhile, the thermostability of the reductase was enhanced by a consensus approach. Such improvements may yield practical catalysts for the asymmetric bioreduction of these aromatic a-keto esters Keywords: aromatic a-keto esters; protein engineering; reductases; substrate specificity Chiral alcohols are frequently required as important intermediates for the introduction of chiral centers into the pharmaceuticals, flavors, aroma and agricultural chemicals, and specialty materials. [1] Enantioselective ketone reduction is a reliable, scalable and straightforward route to optically active alcohols. Biocatalysts are becoming preferred for ketone reduction and the application of reductases in the commercial synthesis of chiral alcohols has undergone a revolution over the past several years. [2] Furthermore, protein en-gineering methods have been applied to generate biocatalysts with higher activity, improved thermostability and/or better selectivity to strengthen the advantages of biocatalysts and to extend their application in the chemical and pharmaceutical industries. [3] Recently, we discovered a versatile keto ester reductase from Candida glabrata (CgKR1), which exhibited a broad substrate spectrum. [4] It showed the highest activity (114 U/mg protein) toward ethyl 4chloro-3-oxobutanoate (COBE, 10, as shown in Figure 2). In contrast, when the substrates were aromatic a-keto esters with a bulky phenyl group, such as methyl ortho-chlorobenzoylformate (CBFM, 1) (Scheme 1) and ethyl 2-oxo-4-phenylbutyrate (OPBE, 4), the activity of CgKR1 decreased obviously by nearly one order of magnitude. In order to enhance the activity of CgKR1 toward the synthesis of these useful chiral alcohols, a rational engineering strategy was adopted to construct NDT libraries [5] of amino acid residues located at the substrate binding site.Given the absence of crystallographic data, threedimensional models of CgKR1 and Gre2p [6] were predicted by homology modeling using the crystal structures of a carbonyl reductase (SsCR) from Sporobolo-Scheme 1. Asymmetric reduction of methyl ortho-chlorobenzoylformate (CBFM) with recombinant cells of E. coli/ pCgKR1 and BmGDH.

Significantly improved thermostability of a reductase CgKR1 from Candida glabrata with a key mutation at Asp 138 for enhancing bioreduction of aromatic α-keto esters

“…The finding in this study is consistent with the widely published theory that the protein surface plays an important role on the protein stability (Bosshart et al, 2013;Jakoblinnert et al, 2013;Martinez et al, 2013). As we hypothesized previously (Huang et al, 2014a), the mutation of I99Y might improve the thermostability of CgKR1 by increasing the hydrogen bonds with water molecules or the hydrophilicity of the enzyme. Substitution of glycine with alanine at position 174 stabilized the enzyme to some extent which may be due to the packing effect or the increased hydrophobic interaction (Johannes et al, 2005).…”

“…One of the bottlenecks for its industrial application is the low thermostability of the wild-type enzyme. Its substrate specificity was shifted from aliphatic keto esters to aromatic esters by two mutations, such as methyl ortho-chlorobenzoylformate (CBFM) and ethyl 2-oxo-4-phenylbutyrate (OPBE), and consensus approach was applied to improve its thermostability (Huang et al, 2014a). Although slight improvement of the thermostability was achieved, further improvement could be expected due to the following reasons.…”

“…Mutant CgKR1-M2 (I99Y/G174A) obtained by consensus approach previously showed better thermostability than the wildtype (Huang et al, 2014a), but the little improvement of the thermostability cannot meet the requirement of its practical application in the bioreduction of CBFM. High biocatalysis loading (15 g/L) would lead to formation of emulsions during downstream processing.…”

“…In order to generate a CgKR1 variant with higher activity toward aromatic ␣-keto esters, such as methyl orthochlorobenzoylformate (CBFM) and ethyl 2-oxo-4-phenylbutyrate (OPBE), the double mutation F92L/F94V which has been shown to be highly active toward CBFM and OPBE (Huang et al, 2014a) was introduced into the variant M5, giving the "hybrid" variant M7 (Fig. 2).…”

Highly efficient enzymatic synthesis of tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate with a mutant alcohol dehydrogenase of Lactobacillus kefir

“…It is known that the substrate specificity of enzymes toward similar substrates always keep in accordance (Huang et al 2014). In the current study, a tetrad mutant of LkADH (LkTADH; i.e., A94T/F147L/L199H/A202L), which is a mutant with improved activity and thermal stability toward a β-ketoester MBO (Campopiano et al 2011), was introduced to evaluate its catalytic efficiency toward CDOH.…”