A recombinant L-threonine aldolase with high diastereoselectivity in the synthesis of L-threo-dihydroxyphenylserine

Abstract

• A new L-threonine aldolase was expressed and mutated. • R318 L showed by far the best stereoselectivity in the synthesis of L- threo -DOPS. • The molecular mechanism for enhanced stereoselectivity was speculated. L-Threonine aldolase (L-TA) is a promising candidate for the optical synthesis of β-hydroxy-α-L-amino acids from glycine and kinds of aldehydes. Because of the C β -stereoselectivity problem, it was usually difficult to optically synthesize β-hydroxy-α-L-amino acids with C β -aromatic groups by application of L-TAs, especially those existing electron-donating groups on C β -aromatic groups, such as anti-Parkinson’s disease drug L- threo -DOPS (L- threo -3,4- dihydroxyphenylserine). A new L-TA (named Sz-1−2) from the gut microbiota of black bears was expressed and its Arg318 site-saturation mutation was conducted. Under the optimized conditions, three mutants R318 M, R318 V and R318 L showed 77.1 %, 79.9 % and 84.9 % de values respectively, which significantly improved than the wild-type (31.2 % de ). Importantly, the mutant R318 L provided by far the best diastereoselectivity in the synthesis of L- threo -DOPS, 1.5 folds higher than the best-ever enzyme (55.4 % de ). The L-TA R318 L is a valuable enzyme for the synthesis of L- threo -DOPS and might also be promising for the enzymatic synthesis of other important β-hydroxy-α-L-amino acids. Moreover, our research also suggested Arg318 was a crucial residue for the improvement of the diastereoselectivity of L-TAs, which could provide valuable experience for the relevant studies.