Semi-rational Design Of TbSADH In The Synthesis Of An Important Medical Intermediate(S)-2-(4-chlorophenyl)pyridylmethanol

(S)-2-?4-chlorophenyl?pyridylmethanol is an important pharmaceutical intermediate,which can be used to synthesize antihistamines such as carbamazine and benzenesulfonate.Now it is mainly by the chemical synthesis with serious pollution and high protection request compared to biocatalysis.Although biological methods are being studied,the catalytic efficiency and stereoselectivity are far from meeting the requirements for drug synthesis.Therefore we adopted semi-rational design methods to modify alcohol dehydrogenase from Thermoanaerobacter brockii?TbSADH?in this research.Based on the crystal structure of TbSADH,the residues lining the binding pocket were included to monitor the structure flexibility using MD simulations.Then the potential candidates for mutagenesis were subjected to CAST/ISM and led to the synthesis of high ee values of chiral alcohol.Meanwhile,the correlation between binding pocket reshaping and catalytic efficiency as well as stereoselectivity was also analyzed to provide theoretical and practical experience for the researches with the same type of catalytic reaction and enzyme engineering.The concrete contents included:?1?In order to monitor the structure flexibility of the residues lining the binding pocket,100 ns MD simulations for the WT enzyme were performed at 30 and 60°C respectively based on the crystal structure of TbSADH?PDB code 1YKF?.The analysis of the root mean square fluctuations?RMSFs?allowed us to identify the flexible regions representing more than 1?fluctuation.The four residues A85,I86,L294,and C295 were relatively rigid,indicating that the less flexible residues may impose restrictions on substrate recognition.To understand how possible mutations of the key residues manipulate pocket fluctuation,the residues were in silico substituted with alanine in terms of alanine scanning,whereas A85 was replaced by glycine.Then 100 ns MD simulations were carried out for the four variants,respectively.As a result,the RMSFs of A85G and I86A showed clear changes compared to the WT.In contrast,L294A and C295A did not show any distinct fluctuations,indicating that substitutions on A85 and I86 are more likely to trigger conformational dynamics.?2?Firstly,the residues A85 and I86 were subjected to saturation mutation based on the structure analysis and MD simulations.As a result,a series of single mutants with low activity were obtained.In order to optimize enantioselectivity and improve activity,a highly focused library combining A85 and I86 with reduced alphabets was generated using the combinatorial active-site saturation test?CAST?concept.Due to steric hindrance of the substrate,only the amino acid alphabets with small steric hindrance?including A,S,T,L,I,V,G and C?were used as building blocks to perform combinatorial mutagenesis at the two positions simultaneously.Eventually,a series of mutants were obtained,which the best mutant T15?A85G/I86L?can produce?S?-enantiomer with the optical purity of 99%e.e.and the yield of98%.Then kinetic parameters,thermostability,preparative scale reaction as well as substrate scope investigation were determined.The results showed that the best mutant T15 had excellent catalytic potential with a k_cat/K_m of 703.52 s^-1?mM^-1.In a higher concentration of substrate(100 mmol?L^-1)in 100 mL of reaction volume,full conversion was achieved within4 h,which maintaining the originally evolved high enantioselectivity of 99%e.e.with 94%isolated yield.It also has excellent thermostability and good activity for most of the ketone substrates.The volume of the binding pocket was expanded to 213±41?³,compared to141±33?³ of WT through MD simulations analysis.Such noticeable differences are responsible for enhancing loop fluctuation by the double mutations?A85G/I86L?.?3?To explore the effect of other residues on enzyme activity and stereoselectivity,the residues,including Q101,W110,L294 and C295 were introduced into the?S?-specific template T13?A85G/I86A?using alanine?A?and serine?S?as the building blocks.The best mutant T33?A85G/I86A/Q101A?can produce?S?-enantiomer with the optical purity of 99%e.e.and the yield of 98%.Then TTN and substrate scope investigation of best mutants were determined and the results showed that T33 had excellent catalytic activity with a TTN of6555 and had good activity for other ketones.In addition,the?R?-specific mutant I86P with poor enzyme activity and stereoselectivity was used as the template to introduce S39,Q101,W110,L294 and C295 for saturation mutagenesis.The best?R?-specific mutant showed the56%conversion?78%e.e.?,and the 71%conversion?70%e.e.?for?S?-specific.