Computational Analysis On Enzyme And Nicotinamide Coenzyme Binding Discipline And Computational Design Of Nictoinamide Coenzyme Specificity Alteration For Enzyme

Nicotinamide coenzymes have attracted great research interests owning to its wide distribution and existence in oxidoreductases. More and more scientific research and practical production have revealed that in the long-term evolution oxidoreductase has shown serious preference to nicotinamide coenzyme I and coenzyme II, which has brought many adverse effects for scientific research as well as manufacturing. This study intends to explore the nicotinamide coenzyme binding discipline with the purpose of coenzyme specificity alteration by means of computer simulation. The main research results of this study include functional exploration of conserved catalytic residues that are related to coenzyme binding and computer-aided coenzyme specificity alteration from NADPH-dependent to NADH-compatible. The major objects involved in this study are all oxidoreductases derived from Gluconobacter oxydans, including Gox0644, Gox2181and Gox0502.The oxidoreductase Gox0644from Gluconobacter oxydans belongs to the NAD(P)-dependent aldehyde keto reductase superfamily. Previous research has found that it includes a highly conserved catalytic tetrad Asp-Tyr-Lys-His, but it is unknown yet what role Asp is playing in catalysis. Based on the crystal structures of wild-type Gox0644and its mutant D53A, this work has designed a set of comparison experiments and utilized molecular dynamics approaches to investigate the potential functions that Asp may have played in catalysis. After analyzing the molecular dynamics trajectory results, we have found that the molecular dynamics trajectory of coenzyme NADPH has shown distinct change in mutant D53A compared to that of wild-type. And when D53A is reverse mutated to A53D, the trajectory of coenzyme NADPH has gradually returned to the situation which is similar to that of wild-type. Thus, it is probable that Asp53may have played an important role in affecting coenzyme binding in the catalytic tetrad. The results of this study may provide a reference and supplement to aldehyde keto reductase superfamily catalytic mechanism study.The oxidoreductase Gox2181from Gluconobacter oxydans belongs to the short-chain dehydrogenas/reductase superfamily. Its feature of oxidizing sugars and polyols incompletely to corresponding materials has attributed its potential industrial applications. Gox2181shows strict cofactor preference of NAD+/NADH in oxidation and reduction of secondary alcohols/ketones. No activities are observed when NADP+/NADPH are used as the coenzyme. Here, we present our novel strategy to engineer Gox2181mutants, which display dual cofactor specificity to NAD+/NADH and NADP+/NADPH. Structure-guided Gox2181mutants have been designed and assessed for stability by SDM and MUpro services online. Subsequently, molecular dynamic simulations were performed to evaluate the compatibility between Gox2181mutants and bound NADP+/NADPH. To this end, we were able to select five Gox2181mutants (Gox2181-D43Q, Gox2181-Q20R&D43Q, Gox2181-D43S, Gox2181-Q20K&D43S and Gox2181-Q20R&D43S) demonstrating high oxidation/reduction activities on secondary alcohols/ketones using both NAD+/NADH and NADP+/NADPH as cofactors. Therefore, our novel computational strategy analyzing the structural re-arrangement of the cofactor binding pocket of the target protein, induced by mutations, should provide an insightful approach to specifically alter the cofactor preference of the target enzymes.The Gox0502from Gluconobacter oxydans is an NADPH-dependent oxidoreductase related to flavin. It has demonstrated great scientific and application value due to its wide substrate spectrum and critical stereospecificity in catalysis. To realize the coenzyme recycling utilization of Gox0502in scientific research and industrial manufacturing, we have engineered Gox0502by site-directed mutagenesis with computer-aided design and made it become an enzyme that can use NADH as its coenzyme. Both results of molecular dynamics trajectory and catalytic activity have indicated that the mutant protein W66Q of Gox0502can utilize NADPH and NADH as well as its coenzymes with comparable catalytic activities. This work has successfully reconstructed the coenzyme specificity of enzyme from NADPH-dependent to NADH-compatible by means of computational protein design.Relying on the disciplinary studies of nicotinamide coenzyme binding and computational simulation approaches, we have effectively realized the coenzyme specificity alteration from NADH-dependent to NADPH-compatible and from NADPH-dependent to NADH-compatible as well. This study should provide strong reference and support to future studies and applications in the fields of synthetic biology, coenzyme engineering and coenzyme related industrial manufacturing.