Discovery And Engineering Of Carbonyl Reductases

Enantiomerically pure chiral alcohols are one of the most important building blocks for production of chiral pharmaceuticals, fine chemicals and agrochemicals. Biocatalytic asymmetric reduction of prochiral ketones is a green and preferred method for the synthesis of chiral alcohols due to its high theoretical yields, remarkable stereoselectivity, few by-products and mild reaction conditions. However, most of ketone-reducing enzymes generally follow the Prelog’s rule in terms of stereochemical outcomes. Biocatalysts with excellent anti-Prelog stereoselectivity are relatively rare in nature. Therefore, this research aims to develop novel biocatalysts with robust characteristics for the synthesis of anti-Prelog chiral alcohols. To that end, screening of ideal microorganisms, gene mining, protein expression, characterization of enzyme catalytic properties and rational design of enzymes were performed.A bacterial strain ZJUY-1401 with anti-Prelog stereospecificity was isolated from soil by an enrichment culture procedure. Based on the colony and microscopic morphology, physiological tests, and 16S rDNA sequence, the isolate was identified as Empedobacter brevis. The whole cells of E. brevis ZJUY-1401 exhibited excellent anti-Prelog stereospecificity toward acetophenone derivatives. The activity and stereoselectivity of the biocatalyst toward acetophenone were significantly increased in the presence of ethanol as cosubstrate. This isolate has advantages such as utilization of cheaper coenzyme, noticeable tolerance against ethanol and remarkable pH adaptability, which make it a promising biocatalyst for the preparation of anti-Prelog chiral alcohols.The carbonyl reductase genes Ebsdr8 and Ppysdr were discovered and cloned from the genome of E. brevis ZJUY-1401 and Pseudomonas putida ATCC 12633, respectively. And the corresponding proteins EbSDR8 and PpYSDR were heterologously expressed in E. coli BL21(DE3). The sequence alignment revealed that both enzymes harbored the highly conserved amino acid residues for all typically short- chain dehydrogenases/reductases (SDRs). The coenzyme NAD(P)H binding motif GXXXGXG was conserved in the N-terminal region, which is the glycine-rich "fingerprints" sequence of the SDR superfamily. The conserved catalytic triad (Ser-Tyr-Lys) was also identified with the active site motif YXXXK. EbSDR8 is capable of catalyzing the reduction of acetophenone to (R)-l-phenylethanol with ee value of 99.9%, while PpYSDR enzyme produced (S)-l-phenylethanol in 84.2% ee. To better understand the relationship between catalytic properties and enzyme structure, the tertiary structural models of PpYSDR and EbSDR8 were predicted by homology modelling. In both model structures, there was an α/β fold with characteristic Rossmann-fold motifs in the active site, showing similar structural features to those of other members in the SDR family. Quality assessment of the resulting models suggested satisfactory accuracy for further structure-based analysis.The recombinant proteins EbSDR8 and PpYSDR were characterized for their biocatalytic properties. The purified EbSDR8 and PpYSDR showed redox activity with either NADH or NADPH as the coenzyme, indicating that the SDRs were not completely specific to its cofactors. Meanwhile, the catalytic performance of EbSDR8 and PpYSDR reduction using NADH was better than that using its phosphorylated counterpart. On the other hand, the activities of both enzymes toward acetophenone were significantly increased in the presence of isopropanol as cosubstrate for in situ cofactor regeneration. The optimum temperature for EbSDR8 and PpYSDR were 35 ℃ and 30 ℃, respectively. And the optimum pH was 7.5 for both of enzymes. Remarkably, EbSDR8 could efficiently catalyze the reduction of prochiral ketones in a broad pH range of 7.0 to 10.5. Biocatalysts with such remarkable pH adaptability are still rare, although it is an important property concerning the application of biocatalysts. Moreover, EbSDR8 and PpYSDR displayed good activity and excellent stereoselectivity toward a range of acetophenone derivatives. EbSDR8 was found to catalyze asymmetric reduction of prochiral aryl ketones to the corresponding alcohols in anti-Prelog configuration, while PpYSDR exhibited opposite stereoselectivity. Based on the structural models and docking results, the geometry and size of substrate binding pocket which regulate the binding orientation and conformation of substrate were considered to play critical roles in determining the stereoselectivity of these two enzymes.Based on enzyme-substrate docking studies and structural comparison of PpYSDR and EbSDR8, strategies involving the decrease of steric hindrance and the introduction/elimination of enzyme-substrate interactions were proposed for the inversion of PpYSDR stereoselectivity. As expected, the structure-guided rational design successfully switched the stereoselectivity of PpYSDR towards halogenated acetophenones from Prelog to anti-Prelog. In particular, mutants M85T/L136E and M85V/M187D reversed the stereoselectivity towards 3,5-bis(trifluoromethyl) acetophenone giving the (R)-product with >95% ee. Excellent anti-Prelog stereoselectivity was also found in the reduction of 2,2,2-trifluoroacetophenone by mutants M85T/W182V and M85V/L136V, which produced (S)-alcohol with ee values of 99.5% and 97.3%, respectively. Enzyme-substrate docking studies indicated that the strategy of introducing enzyme-substrate interactions involving an aromatic ring and a halogen atom was efficient in the control of enzymatic stereoselectivity besides the elimination of steric repulsion. Those results provide valuable insight into how residues involved in substrate binding affect the orientation of bound substrate and thus control the reduction stereoselectivity. This also offers a promising way to expand the sources of anti-Prelog enzymes.Based on enzyme-substrate docking studies, the residues with possible contributions to the differences in substrate-binding pocket were selected as the engineering targets for improving the catalytic activity of EbSDR8. A series of variants were rationally designed and asymmetric reduction of acetophenone derivatives was conducted using these mutants. The designed mutations G94A and S153L indeed enhanced the enzyme activity, and the combination of G94A and S153L resulted in an obviously synergistic effect. The double-mutated variant (G94A/S153L) exhibited a further increased activity toward most of substrates. Particularly, the variant G94A/S153L exhibited obviously improved catalytic efficiency toward acetophenone and 4’-methoxyacetophenone, with up to> 15-fold greater kcat/Km values relative to the wild type, respectively. To gain insight into the molecular basis leading to such a dramatic increase of activity in the reduction of acetophenone derivatives, docking of substrates into the structures of mutant G94A/S153L and molecular dynamics simulations were performed. It was found that the increased steric hindrance and the C—H…π interaction involving aromatic ring of substrate and side-chain alkyl of substituted residues contributed significantly to the enhancement of activity. To evaluate the effectiveness of mutant G94A/S153L, whole-cell-catalyzed asymmetric reduction of 2,2,2-trifluoroacetophenone was performed with isopropanol as the cosubstrate. The recombinant whole cells could effectively catalyze the reduction of high-concentration substrate without addition of any cofactor, giving (S)-2,2,2-trifluoro-l-phenyl-ethanol as product (99% ee), which is an intermediate for several pharmaceuticals applicable for treatment of diseases caused by activation of microglia.