Design of an activity and stability improved carbonyl reductase from Candida parapsilosis

Jakoblinnert, Andre; Wittenboer, Anne van den; Shivange, Amol V.; Bocola, Marco; Heffele, Lora; Ansorge‐Schumacher, Marion B.; Schwaneberg, Ulrich

doi:10.1016/j.jbiotec.2013.02.006

Cited by 37 publications

(19 citation statements)

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“…Stabilization of loops can be achieved by engineering the residues in loops (Daniel et al, 2008;Erwin et al, 1990). 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.…”

Section: Discussionsupporting

confidence: 90%

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

Huang

2015

Journal of Biotechnology

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Section: Discussionsupporting

confidence: 90%

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

Huang

2015

Journal of Biotechnology

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“…The expanded cavity enabling the accommodation of the phenyl group may be comparatively large for the smaller aliphatic group resulting in its lower affinity ( Figure 4C, D). [16] I99Y may increase the hydrogen bonds with water, or increase the hydrophilicity of the enzyme. It suggests the different location of the ester groups of these two substrates, which gives an explanation to the much more favorable binding of the enzyme with the aromatic substrates after opening up the small binding pocket.…”

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confidence: 99%

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

Huang

et al. 2014

Adv Synth Catal

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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.

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“…Obviously, the thermal stability of Ac CR was not very satisfactory. Protein engineering strategy might be promising approach to further improve the activity and stability of the enzyme Ac CR [21].…”

Section: Resultsmentioning

confidence: 99%

A Novel Carbonyl Reductase with Anti-Prelog Stereospecificity from Acetobacter sp. CCTCC M209061: Purification and Characterization

et al. 2014

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A novel carbonyl reductase (AcCR) catalyzing the asymmetric reduction of ketones to enantiopure alcohols with anti-Prelog stereoselectivity was found in Acetobacter sp. CCTCC M209061 and enriched 27.5-fold with an overall yield of 0.4% by purification. The enzyme showed a homotetrameric structure with an apparent molecular mass of 104 kDa and each subunit of 27 kDa. The gene sequence of AcCR was cloned and sequenced, and a 762 bp gene fragment was obtained. Either NAD(H) or NADP(H) can be used as coenzyme. For the reduction of 4′-chloroacetophenone, the Km value for NADH was around 25-fold greater than that for NADPH (0.66 mM vs 0.026 mM), showing that AcCR preferred NADPH over NADH. However, when NADH was used as cofactor, the response of AcCR activity to increasing concentration of 4′-chloroacetophenone was clearly sigmoidal with a Hill coefficient of 3.1, suggesting that the enzyme might possess four substrate-binding sites cooperating with each other The Vmax value for NADH-linked reduction was higher than that for NADPH-linked reduction (0.21 mM/min vs 0.17 mM/min). For the oxidation of isopropanol, the similar enzymological properties of AcCR were found using NAD+ or NADP+ as cofactor. Furthermore, a broad range of ketones such as aryl ketones, α-ketoesters and aliphatic ketones could be enantioselectively reduced into the corresponding chiral alcohols by this enzyme with high activity.

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Design of an activity and stability improved carbonyl reductase from Candida parapsilosis

Cited by 37 publications

References 73 publications

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

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

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

A Novel Carbonyl Reductase with Anti-Prelog Stereospecificity from Acetobacter sp. CCTCC M209061: Purification and Characterization

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