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, Lei; Xu, Jian‐He; Yu, Hui‐Lei

doi:10.1016/j.jbiotec.2015.02.035

Cited by 20 publications

(5 citation statements)

References 54 publications

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“…Replacing amino acid residues at certain positions with the most prevalent ones often result in highly beneficial energy improvements stabilizing proteins [ [67] , [68] , [69] , [70] ]. Huang et al demonstrated that by using consensus approach it was possible to improve the stability of the reductase CgKR1 T 50 15 (temperature at which the enzyme activity is halved within 15 min) by >10 °C [ 71 ].…”

Section: Introductionmentioning

confidence: 99%

FoldX as Protein Engineering Tool: Better Than Random Based Approaches?

Buß

Rudat

Ochsenreither

2018

Computational and Structural Biotechnology Journal

200

125

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Improving protein stability is an important goal for basic research as well as for clinical and industrial applications but no commonly accepted and widely used strategy for efficient engineering is known. Beside random approaches like error prone PCR or physical techniques to stabilize proteins, e.g. by immobilization, in silico approaches are gaining more attention to apply target-oriented mutagenesis. In this review different algorithms for the prediction of beneficial mutation sites to enhance protein stability are summarized and the advantages and disadvantages of FoldX are highlighted. The question whether the prediction of mutation sites by the algorithm FoldX is more accurate than random based approaches is addressed.

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Section: Introductionmentioning

confidence: 99%

FoldX as Protein Engineering Tool: Better Than Random Based Approaches?

Buß

Rudat

Ochsenreither

2018

Computational and Structural Biotechnology Journal

200

125

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“…The EnzymeMiner platform, a user-friendly bioinformatic tool, was employed to retrieve thermophilic enzymes sharing a certain degree of identity with Cp KR. , Using the sequence of Cp KR as a query and identifying Ser133, Tyr171, and Lys175 as the essential residues for catalysis and cofactor binding, four proteins from thermophiles were obtained (Table S1). These four thermophile-originating proteins were then selected for multiple sequence alignment, supplementing with two additional proteins, Ss CR (PDB ID: 5GMO) and Cg KR (PDB ID: 5B6K), , due to their high identities (48 and 46%) and superior stabilities as compared with Cp KR. Based on the assumption that amino acids with the most frequently occurring at a certain position in homologous sequences could contribute more to protein stability than the rarely occurring residues at the same site, 31 mutants were constructed by replacing the targeted residues with more conserved amino acids according to multiple sequence alignment (Figure S1).…”

Section: Resultsmentioning

confidence: 99%

Enhanced Thermostability of Candida Ketoreductase by Computation-Based Cross-Regional Combinatorial Mutagenesis

et al. 2023

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The flexible regions represented by loops have been the focus of protein stability engineering. However, residues located in specific rigid regions are also crucial for protein stability. The role of dynamic correlations between mutations in these two regions on protein stabilization remains inadequately understood. In this work, a ketoreductase CpKR originated from Candida parapsilosis was rationally engineered to improve its stability by comprehensively redesigning the flexible and rigid regions. Hot spots were successively identified and validated in the rigid regions with low B-factor or root-mean-square fluctuation values, as well as in relatively flexible areas of the protein. Based on the non-additive and cooperative mutational effects, a combinatorial variant (M2) with beneficial mutations in these two distinct regions showed a 4630-fold increased half-life at 40 °C and a 12 °C higher apparent melting temperature as compared with those of the wild type. The variant M2 also showed improved pH compatibility, better organic substance tolerance, and enhanced performance in asymmetric reduction of methyl 2-chloro-3-(4-methoxyphenyl)-3-oxo-propanoate (1a), the precursor of cardiovascular drug Diltiazem. The crystal structures and molecular dynamics simulations demonstrated that additional interaction networks in both the rigid and flexible regions modulated protein stability, and the dynamic correlation between these two regions mediated the observed synergistic combination. These results showed that both the flexible and rigid regions, especially the cross-correlated areas, are crucial for enhancing protein stability, and comprehensively redesigning these two regions is a powerful and attractive approach for acquiring stable enzymes.

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“…Cg31 is NADPH dependent and shares a sequence similarity of 78% with Cg KR1, a well-studied KRED which is also originated from C. glabrata. , However, Cg KR1 could not catalyze the oxidation of alcohols such as IPA. Considering its dual activities, Cg31 was designated as Cg ADH.…”

Section: Resultsmentioning

confidence: 99%

Novel Alcohol Dehydrogenase CgADH from Candida glabrata for Stereocomplementary Reduction of Bulky–Bulky Ketones Featuring Self-Sufficient NADPH Regeneration

Sun

Zhang

Yin

et al. 2022

ACS Sustainable Chem. Eng.

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Bioreduction of ketones with self-sufficient cofactor regeneration is green and sustainable for the synthesis of chiral secondary alcohols. In this study, a novel alcohol dehydrogenase CgADH was identified from Candida glabrata through genome hunting, exhibiting high oxidation and reduction activities. Conserved motif analysis revealed that CgADH belongs to the extended SDR subfamily. CgADH is NADP(H) dependent and displays the highest activity at pH 5.0 and 65 °C. Substrate spectrum analysis indicated that CgADH exhibited high specificity toward 2-propanol and 2,3butanediol. Rational engineering of CgADH was performed to modulate its stereoselectivity in the asymmetric reduction of bulky−bulky ketones. Two stereocomplementary mutants, C244A and V222G/C244N, were obtained with e.e. values of 99.6% (R) or 94.5% (S) toward (4-chlorophenyl)-pyridin-2-ylmethanone, respectively. Moreover, the catalytic efficiency (k cat /K M ) of C244A and V222G/C244N increased by 70-and 25-fold compared with the wild type (WT), respectively. C244A and V222G/ C244N also displayed significantly enhanced catalytic efficiency and stereoselectivity toward diaryl ketones with different substituents. Using isopropanol both as a co-substrate and as a co-solvent, a concise selfsufficient NADPH regeneration system was developed to demonstrate the advantage of CgADH. This study provides evidence of the application potential of the newly identified CgADH in the preparation of enantiopure diaryl alcohols with low byproducts and E factor.

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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

Cited by 20 publications

References 54 publications

FoldX as Protein Engineering Tool: Better Than Random Based Approaches?

FoldX as Protein Engineering Tool: Better Than Random Based Approaches?

Enhanced Thermostability of Candida Ketoreductase by Computation-Based Cross-Regional Combinatorial Mutagenesis

Novel Alcohol Dehydrogenase CgADH from Candida glabrata for Stereocomplementary Reduction of Bulky–Bulky Ketones Featuring Self-Sufficient NADPH Regeneration

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