Mining,Engineering,Crystal Structures And Catalytic Mechanisms Of D-carbamoylases

D-amino acids are important amino acids and are widely used in pharmaceutical,food and agriculture industries.The hydantoinase process is a three-enzyme cascade reaction including hydantoin racemase,hydantoinase and carbamoylase.It is an economical and environmentally friendly method for producing optically pure amino acids.Whereas,the low enzyme activity and poor stability of D-carbamoylase can limit its industrial application.To obtain D-carbamoylase with high enzyme activity and thermostability,two D-carbamoylases from Arthrobacter crystallopoietes CGMCC 1.1926 and Nitratireductor indicus CGMCC 1.10953,AcHyuC and NiHyuC,were identified by genome mining.Both AcHyuC and NiHyuC were evaluated in hydantoinase process.To further improve the catalytic efficiency of D-carbamoylase,NiHyuC with higher k_catvalue and thermostability was selected as the candidate for further protein engineer and catalytic mechanism study.The main conclusions are as follows:（1）Mining and characterization of D-carbamoylases:Specific activity of purified AcHyuC and NiHyuC toward N-carbamyl-D-tryptophan（3a）are 0.84 and 1.94 U·mg^–1,both of which are highly enantioselective and non-metal ion dependent enzyme.The optimum p H and temperature of AcHyuC and NiHyuC are p H 8.5 and p H 8.0,and 30°C and 35°C,respectively.The half-life of AcHyuC and NiHyuC are 12 h and 72 h at 30°C.Both enzymes prefer N-carbamoyl amino acids with larger steric hindrance.AcHyuC and NiHyuC has k_cat values of39.5 and 209.9 min^–1 toward 3a,K_m values of 1.4 and 8.9 m M,and k_cat/K_m values of 30.4 and25.7 min^–1·m M^–1.（2）Construction of hydantoinase process cascade reaction:Cascade reactions of AcHyuC and NiHyuC were constructed by coupling with hydantoin racemase（Aa Hyu A）from Arthrobacter aurescens DSM 3747 and the D-hydantoinase（At Hyu H）from Agrobacterium tumefaciens BQL9.In a 0.5-L system containing 10 k U·L^–1 Aa Hyu A,5 k U·L^–1 At Hyu H and 10k U·L^–1AcHyuC,D-Trp yield of 98.4%and productivity of 36.6 g·L^–1·d^–1 toward 80 m M L-indolylmethylhydantoin（1a）was achieved at 24 h.Cascade reactions of Aa Hyu A/At Hyu H/AcHyuC and Aa Hyu A/At Hyu H/NiHyuC exhibited similar performance when catalyzing 100 m M 1a,resulting yields of 81.4%and 79.1%at 24 h,respectively.Therefore,NiHyuC with higher activity and thermostability was selected as the candidate for further protein engineering.（3）Crystallization and protein engineering of NiHyuC:Crystal structure of WT（2.80?）was obtained by crystallization.Both random mutagenesis and structure-guided design were used to engineering NiHyuC.Nine single variants were obtained and the best variant M4（A200N/D187N/S207A/R211G）was obtained by combinatorial mutagenesis,which exhibited a k_cat/K_m value of 1135.0 min^–1·m M^–1（44.2 times of WT）,and K_m value of 0.4 m M（4.5%of WT）.When applied in cascade reaction,it can reach a yield of 99.3%and productivity of 64.9g·L^–1·d^–1at 12 h toward 160 m M 1a in 0.5-L scale,with 10 k U·L^–1 Aa Hyu A,5 k U·L^–1 At Hyu H and 60 k U·L^–1M4.（4）Crystallization of NiHyuC variants and elucidation of its catalytic mechanism:Crystal structures of R211G（2.37?）,M3-1（2.70?）and M4（2.14?）were obtained by crystallization to elucidate the evolutionary changes.Based on crystal structure and molecular dynamics simulation,loop 200–207 of NiHyuC was identified to play an important role in modulating access to the substrate entrance tunnel.The enhanced flexibility and outward swing of loop200–207 in M4 could be beneficial for the substrate access to catalytic center.Pre-reaction state and binding free energy computation show that M4-3a is more readily to enters a pre-reaction state and have a lower binding free energy compared with WT,which explains its enhanced k_cat/K_m and decreased K_m values。...