Enzymatic Properties And Crystal Structure Analysis Of Branched-chain Amino Acid Aminotransferase From Pseudomonas Sp.

Aminotransferase(AT)is an important biocatalyst for the synthesis of chiral amines or ketonic acid due to its ability to transfer amino groups into ketones or keto acids and has high enantioselectivity and regioselectivity.In all ATs,branched-chain amino acid aminotransferases(BCATs)reversibly catalyze the cleavage of branched-chain amino acids(BCAAs)such as L-valine,L-leucine and L-isoleucine,with alpha-ketones.Glutaric acid produces the corresponding keto acid and L-glutamic acid.Currently,BCAT has been applied to the biosynthesis of unnatural amino acids and branched keto acids including L-tert-leucine.Despite the enormous potential of BCAT,most enzymes still lack a wide range of substrates and activities,so the study of new sources and novel enzymes has great promise.Here,we explored four different sources of BCAT by used bioinformatics methods,which was cloned,expressed and purificated in Escherichia coli.After the activity comparison,BCAT from pseudomonas(PsBCAT)was selected to further analyze its properties and structure.The results of enzymatic properties showed that the optimum reaction pH,temperature and concentration of PLP of PsBCAT were 8.5,40?and 10mM respectively.PsBCAT exhibits a relatively broad substrate spectrum and has significantly high activity against L-leucine,L-valine,L-isoleucine and L-phenylalanine,with an activity of 105 U/mg,127 U/mg,115 U/mg and 98 U/mg,respectively.Further,PsBCAT is active against aromatic L-amino acids,L-histidine,L-lysine and L-threonine.At the same time,in order to analyze the catalytic mechanism of PsBCAT with a broad substrate spectrum,the crystal structure of PsBCAT was also analyzed.Based on the determined crystal structure,we found that there are some differences in the substrate binding pocket,which may affect the substrate specificity of the enzyme.In addition,in practical applications,coupled with ornithine aminotransferase(OrnAT)from Bacillus subtilis,the coupling system was applied to the preparation of L-tert-leucine with a conversion of 83%.We demonstrated efficient biocatalytic asymmetric synthesis of _L-tert-leucine using a coupling system,which can be used to remove the inhibitory by-product,and to shift the reaction equilibrium towards the product formation.In summary,the structural and functional characteristics of PsBCAT were analyzed in detail.This information will play an important role in the synthesis of chiral amino acids,which is beneficial to the production of ketonic acid and enantiomerically chiral amines by aminotransferase.