Engineering the substrate specificity of oxidoreductases by redesign of enzyme-substrate intermolecular interactions

Abstract

Enzymes are versatile biocatalysts for the application of industrial biotechnology. However, the use of natural enzymes to the broad area of biotechnology can be limited by their finite catalytic repertoire. Engineering the substrate-specificity of enzyme is a promising approach that can expand the applicability of enzymes. So far, rational design and computational design approaches have been applied as useful structure-based design strategies for the engineering of enzyme-substrate specificity. However, due to the problems of the two approaches, such as the insight-dependency in rational design and the complex and non-efficient properties of the algorithms in computational design, an efficient and quantitative design approach is required. On the other hand, the basic mechanism of enzyme catalysis begins with the binding of the substrate to the active site on the enzyme, and the engineering of enzyme-substrate specificity is achieved by enhancing the catalytic reaction rate of the enzyme toward the non-native target substrates. The binding and catalytic characters of enzymes, therefore, are the key factors for the engineering of the enzyme-substrate specificity. In this study, a new design strategy considering both of the substrate binding and catalytic mechanism was proposed for the engineering of enzyme-substrate specificity. This approach excludes the drawbacks while taking the advantages of the traditional approaches. By applying molecular docking simulation, the binding mode of the target substrate was analyzed, and the binding free energy of the substrate was calculated to quantitatively describe the binding affinity of the substrate. In addition, the enzyme-substrate atomic contact analysis was applied for the selection of the mutation sites in the binding pocket of the enzyme. On the other hand, the analysis of catalytic mechanism of the enzyme was applied to consider the catalytic character of the model enzymes. In this study, two oxidoreductases, 3-hydroxybutyrate dehydrogenase (3HBDH) and succinic semialdehyde reductase (AKR7A5), that follow the H- transfer mechanism were selected as model enzymes, and the donor-acceptor distance was employed as key design parameter for the catalytic properties of the oxidoreductases. It is because the donor-acceptor distance has been reported as an important factor in the H- transfer mechanism. In this regard, the overall design strategy was established

to increase of the binding energy and to decrease of the donor-acceptor distances by manipulating the enzyme-substrate intermolecualr interactions. With this approach the use of transition state modeling that requires quantum-mechanical approach could be avoided. In addition, the validity of the strategy can be theoretically supported by the barrier compression model

the reduction of donor-acceptor distance brings about the compression of the activation barrier, thus enhance the catalytic reaction rate. The substrate-specificity of 3HBDH from Alcaligenes faecalis toward levulinic acid was engineered. The enzyme-substrate interatomic contact analysis was applied for the selection of the mutation sites, and the design strategy of increasing binding energy with decreasing average donor-acceptor distance was applied for the generation of the improved variants. The 16 variants of the 3HBDH were generated focusing on the key substrate binding residues, His144 and Trp187, and the positive correlations, that support the feasibility of the strategy, between the design parameters and the experimental kinetic parameters were obtained. Among the variants, a double mutant, His144Leu/Trp187Phe, showed the most enhanced catalytic activity (33.4-fold) toward the target substrate. Approximately 100% conversion of levulinic acid to 4-hydroxyvaleric acid was achieved with this double mutant in 4 hr, while 50% conversion occurred with the wild-type. On the other hand, the structural effect of the positive mutations on the substrate-specificity of the enzyme was also analyzed by employing the interatomic contact analysis to understand the structural basis for the substrate specificity of the enzyme. The substrate-specificity of AKR7A5 with levulinic acid was engineered. The redesign of the enzyme-substrate interatomic contacts was applied for the selection of the mutation sites, and the engineering was performed focusing of the Met13 residue. The binding energy and average donor-acceptor distance were incorporated as design parameters, and four out of the six tested mutants showed improved catalytic properties toward the target substrate. Among the improved variants, Met13Trp exhibited the most enhanced activity (7.0-fold) toward the target substrate. The simulation results were validated with the experimental data, and the relationship between structural and kinetic parameters was investigated. The results supported the feasibility of the design strategy. In addition, the structural effect of the positive mutations on the substrate-specificity of the enzyme was analyzed by employing the interatomic contact analysis to understand the structural basis for the substrate specificity of the enzyme. Finally, overall discussion on the feasibility of the design strategy was performed considering the barrier compression theory. On the other hand, the future directions for a general application of the proposed strategy were suggested considering the activity theory of enzymes including electrostatic preorganization and conformational dynamic effects.