Catalytic Mechanism And Molecular Engineering Of L-isoleucine Dioxygenase And Construction Of Intracellular 2-ketoglutarate Generation System

Fe（Ⅱ）/2-ketoglutarate-dependent dioxygenases（Fe（Ⅱ）/2OG DOs）belong to a diverse proteins superfamily with members from bacteria,fungi,plants,and vertebrates that catalyze a variety of oxidative transformations,including hydroxylation,halogenation,ring formation,desaturation,isomerization,ring expansion,and epoxidation reactions.Currently,these enzymes are mainly used in the biosynthesis of hydroxylated amino acids and antibiotics.However,due to the limited structural information resources of Fe（Ⅱ）/2OG DOs and low sequence homology,the catalytic rules of Fe（Ⅱ）/2OG DOs are not clear yet,and in addition,their catalytic reactions require a continuous supply of the 2-ketoglutarate（2OG）,which limits its application in biosynthesis synthesis.In this thesis,L-isoleucine dioxygenase（IDO）from Bacillus thuringiensis was used as a target for the selective synthesis of chiral hydroxyl amino acids,and the crystal structure of IDO was resolved,and its catalytic molecular mechanism was revealed based on protein structure and molecular dynamics simulation;in view of the substrate specificity of IDO catalyzed aliphatic amino acids,the substrate recognition pattern of IDO catalyzed amino acid hydroxylation was predicted by molecular dynamics,and the substrate spectrum of IDO was expanded by rational modification;the cellular metabolic pathway was modified by multi-gene editing technology,which enhanced the intracellular regeneration efficiency of 2OG and promoted the synthesis of4-Hydroxyisoleucine（4-HIL）product.The main results are as follows:（1）Expression,purification and structural analysis of IDO.The plasmid p ET-28a（+）-ido was transformed into E.coli BL21（DE3）to express IDO protein and was subjected to Ni-Charged Resin FF column chromatography,and concentration in ultrafiltration centrifuge tubes to obtain the IDO protein for crystallization.X-ray diffraction identified the IDO crystal space group as P21 with a resolution of 2.34?.The IDO crystal structure is a homotetramer and each chain structure binds a Fe²⁺atom at the active pocket.The IDO monomer molecule consists of 12β-chains（β1-β12）,4α-helices（α1-α4）and two short 3₁₀helices（η1-η2）with 240amino acids.Mostβchains form two antiparallelβfolds:the large six-strandβfolds（Sheet1;β1,β2,β4,β5,β9,andβ12）and smaller three-strandβfolds（Sheet2;β7,β8,andβ10）.Β3,β11,andβ6 are extended toβ2,β10,andβ7,respectively.These twoβsheets are close to each other to form a cup-shapedβ-sandwich structure with the the topological characteristics of double-chainβ-helix.The helices are on one side of SHEET1,helping to stabilize it.Three flexible loops in the IDO active pocket are located betweenβ3 andα3（L1）,β4 andβ5（L2）,andβ9 andβ10（L3）.（2）The molecular mechanism analysis of amino acid hydroxylation catalyzed by IDO.DALI structure similarity search indicates that IDO structure belongs to the Cupin superfamily protein.The IDO catalytic core is located on the C-terminal residues 134-236,while the N-terminal residues serve stablilize the Cupin core and contribute to the formation of the active site pocket（near L1）.The residues bound to Fe²⁺are strictly conserved between IDO and structural homologues,which indicates that His159,Asp161,and His212 of IDO are involved in Fe²⁺binding.One conserved residue（H212）bound to Fe²⁺is located onβ10,and the other two conserved residues（H159 and D161）are located on L2.The molecular dynamics simulation results of IDO and L-isoleucine（L-Ile）showed that S153 and P155formed hydrogen bond with the amino group of L-Ile,and residues of Y143 and R227 had hydrogen bond with the carboxyl group of L-Ile in the active pockets of IDO.Residues S153,P155,Y143,and R227 form a polar substrate binding pocket,which may be important for substrate recognition and catalytic orientation.The distance between Fe²⁺and C4 of L-Ile may be important for hydroxylation.（3）Substrate specific molecular modification of IDO-catalyzed amino acid hydroxylation.Since IDO has a wide substrate spectrum for L-type aliphatic amino acids,molecular dynamics was used to simulated IDO and the L-type amino acids.The simulation results showed that the nearest position of Fe²⁺is at the C4 position of the amino acid carbon chain,while the distance between the C2 of 2OG and the C4 position of these L-type amino acids is about 4.4?.It is indicated that the distance between Fe²⁺,C2 of 2OG and C4 position of L-type aliphatic amino acids plays an important role in IDO hydroxylation.Based on polar pocket and residue contribution,three mutation sites（Y143,S153 and R227）were selected for single point saturation mutation,and L-type aliphatic amino acids and aromatic amino acids were catalyzed in vitro.The results showed that most of the mutations made the catalytic activity lower than that of the wild-type IDO.Unexpectedly,mutants Y143D,Y143I and S153A showed catalytic activity for L-phenylalanine（Phe）,while mutants Y143I,S153A,S153Q and S153Y exhibitd catalytic L-homophenylalanine（HPhe）activity.The molecular docking showed that the C3 of Phe and the C4 of HPhe are closer to the Fe²⁺and C2 of 2OG,and the corresponding binding free energy is also increased,which is more beneficial to the hydroxylation of C3 of Phe and C4 of HPhe under attack.This was also confirmed by mass spectrometer analysis of the product.This indicates that IDO is expected to be used as the target enzyme to hydroxylate aromatic amino acids and expand its application properties.（4）Construction of 2OG coupling system based on CRISPR-Cas9.The two-plasmid-based CRISPR-Cas9 multigene editing system was constructed and used to efficiently and rapidly knock out the suc AB and ace AK genes in the TCA cycle pathway of E.coli to achieve the accumulation of 2OG,and to reconstiture the TCA cycle to reuse 2OG by expressing ido gene in mutant bacteria,and then improve the transformation efficiency of L-Ile.The results showed that the conversion rate of L-Ile in recombinant strain E.coli BL21（DE3）Δsuc ABΔace AK/p ET 28a（+）-ido was about 15%higher than that in E.coli BL21（DE3）/p ET-28a（+）-ido.This indicates that the efficient and rapid editing strategy of two-plasmid-based CRISPR-Cas9 has an important potential for multi-gene editing and optimization of strains for industrial production.