Gene Mining, Catalytic Performance And Molecular Engineering Of Halohydrin Dehalogenase

Halohydrin dehalogenases?HHDHs?can catalyze the dehalogenation of halogenate compounds and ring opening of epoxides under mild conditions,which shows great application potential in the synthesis of chiral epoxides and?-substituted alcohols.However,up to now,only HheC from A.radiobacter AD1 was found to show remarkable enantioselectivity in the conversion of various halohydrin and epoxides.In order to meet the needs of the application,the development of new biocatalysts with high efficiency,excellent enantioselectivity and high activity has become the focus of basic research on industrial application of HHDHs.Ten genes with 31%-48%identify with HheC and HheB were selected from NCBI database and overexpressed in Escherichia coli?E.coli?.As a result,three HHDHs were identified from nine successfully expressed enzymes with good activity and different enantioselectivity towards the representative substrate?R,S?-phenyl glycidyl ether?PGE?.In addition,it was found that,in general,both N-or C-terminal His6 tags have a noticeable negative effeft on activities of HHDH8 and HHDH9.Therefore,HHDH_His10 from Pseudomonas pohangensis was chosen for further studies that was identified with 44%sequence identify to HheB and 31%sequence identify to HheC and exhibited R selectivity for?R,S?-PGE.The HHDH_His10 was purified to homogeneity by a Ni-NTA column and functionally characterized.The optimal temperature was obtained at 40�C in the ring-closure reaction.The recombinant HHDH_His10 showed maximum activity at pH 7.5 in the ring-opening reaction,while the maximum activity was obtained pH 10.0 in the ring-closure reaction.In addition,the purified HHDH_His10 was highly stable at pH 6.0-7.5 and below 50�C.The purified HHDH_His10 showed a very broad substrate range towards various halohydrins and epoxides.The michaelis constant K_m and V_max values were calculated to be 31.75 mM and 32.79?mol�min^-1�mg^-1 with?R,S?-PGE as the substrate,respectively.The enantioselective biosynthesis of S-PGE from racemic?R,S?-PGE was investigated and the maximum ee values and yield of?S?-PGE reached>99%and 16.35%at 20 mM?R,S?-PGE by recombinant HHDH_His10.Enantiopure?R?-7 could be obtained with>99%ee and about 25%yield at pH 7.5 and 8.0,respectively.To improve the HHDH_His10 potential for producing?S?-PGE,an enhancement of the enantioselectivity toward?R,S?-PGE would be highly desirable.Molecular docking of?R,S?-PGE into the active site of wild-type HHDHHis10 allowed the identification of proximal HHDH_His10 active site residues.Six amino acids residues near the substrate binding pocket?Gln159,Asn160,Phe161,Tyr167,Phe168 and Pro169?were selected as targets for site-saturation mutagenesis and combined mutagenesis.No positive mutant with improved enantioselectivity was isolated at position Phe161,Tyr167 and Phe168.Three mutants Q159L,N160L and P169Q were selected and subsequently shown to have increased enantioselectivity for kinetic resolution of?R,S?-PGE,while their E-value raised from 9.85 to 21.80,21.10 and 10.84.It was worth mentioning that the mutant N160L displayed an inverted enantioselectivity?from R to S?toward?R,S?-PGE.In addition,combination of mutations can have subtractive effects in the case of enantioselectivity.After purification,the enzyme activities of mutants were both decrcased and were 56.75%and 47.45%of HHDH_His10.The results of substrate specificity showed that mutants N160L and Q159L displayed increased enantioselectivity toward PGE and derivatives,but substrate 3-?2-ethylyphenoxy?propane-1-azido-2-ol?12?and styrene oxide?16?were exceptions.Especially,the E was increased from 1.85 to 25.77 for substrate 3-?4-Methyphenoxy?propane-1-azido-2-ol?11?by mutant N160L.A possible mechanism was proposed to explain the experimental observations obtained for the wild-type and mutant HHDH_His10.Molecular docking of both enantiomers of?R,S?-PGE to the wild-type HHDH_His10 and the mutant N160L were performed using program AutoDock.According to the proposed HHDH reaction mechanism,the distance between oxygen atom of epoxide??R?-PGE and?S?-PGE?and two key aminos?Tyr129 and Ser116?plays acrucial role in determining the HHDH enantioselectivity towards the substrate.The enantioselective biosynthesis of?R?-PGE was investigated and the maxmium ee values and yield of?R?-PGE reached>99%and30.92%in the kinetic resolution of racemic PGE by mutant N160L.