Mining And Characterization Of Fe(Ⅱ)/α-ketoglutarate-dependent Dioxygenase And Its Synthesis Of (2S,3R)-3-hydroxypipecolic Acid

Chiral hydroxylated amino acids can be performed as chiral precursors,intermediates and final products,and are widely used in the synthesis of multifunctional biological macromolecules,pharmaceuticals and fine chemicals.Fe(Ⅱ)/α-ketoglutarate(2-OG)-dependent dioxygenase have great advantages over traditional synthetic chemistry in catalyzing C-H functionalization due to their regioselectivity and stereoselectivity.This family of enzymes can catalyze a variety of oxidative transformations,of which the most fully studied reaction is substrate hydroxylation.In this paper,the substrate specificity of Fe(Ⅱ)/2-OG-dependent dioxygenase with hydroxylation function was investigated;the catalytic activity of L-proline cis-3-hydroxylase(cis-P3H)toward the unnatural substrate L-pipecolic acid(L-Pip)was verified;protein engineering was carried out to solve the problem of low efficiency of cis-P3H catalyzing the formation of(2S,3R)-3-hydroxypipecolic acid.The main results are as follows.(1)The substrate specificity was evaluated by measuring the production of by-product succinic acid using the succinic acid kit.The expression verification and enzyme activity detection of twelve candidate enzymes with hydroxylation function were carried out,and the substrate spectrum detection of successfully purified recombinant proteins KaPH1,cis-P3H,VioC,AsnO,AsnOD241N,IDO and NkLH4 were detected.VioC,AsnO,AsnOD241N and NkLH4 had strong substrate specificity and only specifically catalyzed their respective natural amino acid substrates;KaPH1,cis-P3H and IDO showed certain substrate tolerance,KaPH1 and cis-P3H could catalyze L-proline and different ring size or ring-substituted proline analogues,IDO could catalyze L-isoleucine and some hydrophobic aliphatic amino acids.(2S,3R)-3-hydroxypipecolic acid catalyzed by cis-P3H was an important and valuable chiral building block.Therefore,(2S,3R)-3-hydroxypipecolic acid was selected as the target product and cis-P3H as the target enzyme.(2)Protein engineering was modified by structure-oriented semi-rational design.Based on the static structure analysis,the "handlebar" binding mode related to the substrate interaction was proposed,and the force balance affected the stable binding of the substrate,so the mutation hot spots R97 and E112 were selected.At the same time,the dynamic changes of the active pocket sites were investigated by molecular dynamics simulation,and the mutation hotspots L57 and G282 in the loop regions were selected.Aiming at the main problem of low catalytic activity of the target enzyme cis-P3H,site-directed saturation mutagenesis of the above key sites was performed.(3)The construction and screening of mutant library were carried out.R97 was a key site related to improving catalytic activity,and the catalytic efficiency of the dominant mutant R97M was 1.83-fold that of the wild type.By analyzing the substrate binding pocket before and after the mutation,adjusting the balance of "handlebar" and broadening the substrate binding space were the possible reasons for the improvement of R97M catalytic efficiency.The interaction analysis of the key site R97 was carried out to select the second-round of modification hotspots.Six positive mutants were obtained by site-directed saturation mutagenesis of N43 based on R97M.The specific activity of the most dominant mutant R97M/N43T was 0.46 μmol·min-1·mg-1,which was 2.42-fold that of R97M.Structural analysis revealed that R97M/N43T further broadened the substrate binding space.(4)The reaction conditions(temperature and pH)and the concentration of reaction components(Fe2+,2-OG and Vc)of R97M/N43T were optimized under crude enzyme condition.The activity of R97M/N43T was low under high temperature,acidic environment and high concentration of Fe2+.The reaction was not completely dependent on exogenous Fe2+,but dependent on 2-OG.The concentration of 2-OG required for the reaction was excessive than the substrate concentration,and the addition of Vc could promote the reaction.When the concentration of L-Pip was 10 mmol·L-1 and under the optimal conditions(temperature:10℃,pH:7.5(Tris-HCl buffer),Fe2+:0.05 mmol·L-1,2-OG:30 mmol·L-1,Vc:15 mmol·L-1),the molar yield of(2S,3R)-3-hydroxypipecolic acid catalyzed by R97M/N43T was 88%,which was 4.11-fold that of wild type.