Rational Design Of NAD~+-dependent Oxidoreductase Based On Enzyme Catalytic Cycle

NAD+-dependent oxidoreductase has high activity,stereoselectivity and regioselectivity,and is a research and development hotspot for the production of chiral compounds in the pharmaceutical industry,fine and specialty chemicals.Rational design and directed evolution based on sequence and structural information have become the center of enzyme engineering.The thesis focuses on the description of the enzyme catalytic cycle process:substrate migration and recognition,biochemical reaction and product release.Rational design of malate enzyme,alanine dehydrogenase and meso-2,3-butanediol dehydrogenase were carried out,and the results were as follows:(1)The catalytic cycle of malate enzyme(EC 1.1.1.40)was dynamically described in atomic details,and reveal the structural basis of malate enzyme stereoselective substrate recognition.L-malate was the natural substrate of malate enzyme with higher binding energy,while D-malate as a substrate analogue inhibitor had lower binding energy.D-malate showed a coplanar ion reaction with Arg165 and nearly 90° coordination angle with Mn2+.This binding conformation resulted in D-malate with higher affinity and higher activation energy barrier than L-malate.Catalytic mechanism was outlined:?-decarboxylation step was the rate-limiting step with a 16.44 kcal/mol effective activation barrier and about 4.58 kcal/mol higher than hydride transfer.The result was consistent with the experimentally derived kcat values.L/D-malate needed to overcome a higher barrier than pyruvate to break all bonds in parallel and then to escape from the binding pocket.Leu167 and Asn421 comprised a swinging gate to control the product release.The more open gate was required in the direction of pyruvate to L-malate.(2)The catalytic mechanism of alanine dehydrogenase(EC 1.4.1.1)was studied by using QM/MM methods at PM6 level,to improve the activity of alanine dehydrogenase amination reaction for alanine synthesis.We showed that the energy barrier for hydride transfer was 39.71 kcal/mol and the energy barrier for nucleophilic attack of water was about 8 kcal/mol when His96 served as general acid/base.Therefore,hydride transfer was the rate-limiting step and His96,not Lys75 was confirmed to act as the catalytic acid/base.When Tyr94 mutated to Phe94,the three methods of QM/MM-US,ONIOM and QM/MM-SMD were calculated at the theoretical level of PM6,B3LYP/6-31G*and B3LYP/6-31G*,respectively.The results showed that the activation energy barrier of-deamination reaction increased 2.33 kcal/mol,0.9 kcal/mol and 1.67 kcal/mol,the kcat value of deamination reaction would decrease.Activation energy barrier of amination reaction decreased 3.29 kcal/mol,1.0 kcal/mol and 1.18 kcal/mol,the kat value of ammoniation reaction would increase.The MM/PBSA method combined with the interaction entropy method predicted that the binding free energy of the deamination reaction would decrease by 3.4 kcal/mol,Km value would decrease.In the ammoniation reaction,the binding free energy increased slightly when the binding free energy increased by 0.89 kcal/mol,Km would increase slightly.Recombinant expression of Tyr94Phe mutant,enzyme kinetic parameters showed that the kcat value of the deamination reaction was reduced by 56%,the kcat value of the amination reaction was increased by 30%,and the destabilization of the ground state caused the Km value of the amination reaction to increase by 30%.(3)The active tunnel of meso-2,3-butanediol dehydrogenase was identified by molecular dynamics simulation.The two short ?-helixes positioned away from the ?4-helix possibly expose the hydrophobic ligand-binding cavity,gating the exit of product and cofactor from the activity pocket.Further MM/GBSA binding free energy analysis showed that Phe212 and Asn146 function as the key product-release sites.Site-directed mutagenesis experiments targeted to the sites showed that the original activity of Asn146Gln was retained,but the activity of Asn146Ala mutation was lost and the kcat of Phe212Tyr was enhanced up to(3-7)-fold that of wild type,and the catalytic efficiency was enhanced to 2-4 times of wild type.(4)The binding free energy was evaluated by the number of the productive poses,MM/PBSA-interaction entropy and thermodynamic integration,and the activation barrier was evaluated by ONIOM and QM/MM-SMD at the theoretical level of B3LYP/6-31G*.Our simulation results proved that the main factor determining the stereoselectivity of meso-2,3-butanediol dehydrogenase was substrate binding,the binding free energy of the natural substrate(3R)-acetoin was lower than(3S)-acetoin.The main factor that determined stereoselectivity of(2S,3S)-2,3-butanediol dchydrogenase was catalytic process,the energy barrier for reducing the natural substrate(3S)-acetoin was lower than the energy barrier for reducing(3R)-acetoin by 2.52 kcal/mol.2,3-butanediol dehydrogenase catalyzed the formation of acetoin from diacetyl to be an irreversible reaction.The main reason was that acetoin couldn't be stably bound in the active pocket.The recombinant protein with site-directed mutations,and enzyme activity analysis proved that Trp190Met has acetoin oxidation activity.Using the site of Trp190Met as a reference,a 2,3-butanediol dehydrogenase from Lactococcus lactis(Ll-BDH)was found which catalyzed the oxidation of acetoin to diacetyl.Km was 283.70 mM and kcat was 0.49 s-1,the reduction of diacetyl to acetoin was proved to be a reversible process.Lli-BDH was the first reported enzyme that oxidized acetoin to form diacetyl.In summary,global simulation of enzyme catalytic cycle revealed the mechanism of enzyme catalysis at the atomic level,expanded the knowledge of enzyme structure and function,the stereoselective mechanism of malate enzyme and meso-2,3-butanediol dehydrogenase were elucidated.By destabilizing the ground state,the kcat value of the amination reaction of alanine dehydrogenase was increased;by modifying the product release channel,the catalytic efficiency of meso-2,3-butanediol dehydrogenase was improved;and the probability of forming reactive conformation was increased based on substrate binding,an enzyme with the activity of oxidizing acetoin to diacetyl was designed and reported for the first time.The established NAD+-dependent oxidoreductase rational design technology based on enzyme catalytic cycle,combining high throughput experimentation,advanced molecular biology techniques and machine learning methods to quickly obtain enzymes with a variety of natural and even unnatural catalytic activities,make full use of low-cost and more sustainable biocatalysis to promote the industrialization of biocatalysis.