Coenzyme Alteration Of Malate Dehydrogenase And Its Physiological Roles In Escherichia Coli

Malate dehydrogenases （MDH, EC1.1.1.37） catalyze the reversibleconversion between L-malate and oxaloacetate. MDHs participate in thetricarboxylic acid cycle （TCA）, glyoxylate cycle, malate-asparate shuttle, thefixation of CO₂in photosynthesis and other important metabolic pathways in thecell. Phylogenetic analysis indicates that MDHs can be divided into three groups:cytoplasmic homodimer MDHs, mitochondrial homodimer MDHs andhomotetramer lactate dehydrogenase （LDH）-like-MDHs.According to the difference of coenzyme specificity, MDHs can be classifiedinto two types: NAD⁺-dependent MDH （NAD-MDH） and NADP⁺-dependent MDH（NADP-MDH）. Current studies show that most MDHs are NAD⁺dependence withthe exception of NADP-MDHs from plant chloroplast and some archaea. Despitethe stereochemical structure similarity between NAD⁺and NADP⁺, theirphysiological roles are dramatically different: NADH participates in the catabolismand provides the energy for cell growth, while NADPH involves in the anabolismand is used as reducing power for biosynthesis. Therefore, MDHs with differentcoenzyme specificity have different functions.On the basis of amino acid sequence and3D structure alignments betweenNAD-MDH and NADP-MDH, amino acid residues involved in NAD⁺-binding inEscherichia coli MDH （EcMDH） were substituted with the corresponding residuesin NADP-MDH by site-direct mutagenesis. Three mutants (G³⁴S³⁵S³⁶G⁷⁷,G³⁴S³⁵S³⁶G⁷⁷A⁷⁹and G³⁴S³⁵S³⁶G⁷⁷G⁷⁹) were constructed accordingly. Kineticanalysis of the wild-type EcMDH and three mutants showed that the coenzymespecificities of three mutants were successfully converted from NAD（H） toNADP（H）. The preference to NADPH of the mutant G³⁴S³⁵S³⁶G⁷⁷A⁷⁹was about28-fold to NADH, and also about4.1×10⁴-fold than that of wild-type EcMDH.In order to investigate the effect of MDH with different coenzyme specificityon the cell growth, the NAD-MDH genes on the chromosome of wild-type E. colistrain Wt (icdA^NADP/mdh^NAD) carrying the NADP-dependent IDH and engineering E. coli strain Ym (icdANAD/mdh^NAD) harboring the engineered NAD-dependent IDHwere substituted with the mutated NADP-MDH gene encoding mutantG³⁴S³⁵S³⁶G⁷⁷A⁷⁹by gene knock-out and knock-in methods. Two mutant strainsWt/mdh (icdA^NADP/mdh^NADP) and Ym/mdh (icdANAD/mdh^NADP) were constructed,and the growth rates of four strains Wt (icdA^NADP/mdh^NAD), Ym (icdANAD/mdh^NAD),Wt/mdh (icdA^NADP/mdh^NADP) and Ym/mdh (icdANAD/mdh^NADP) were measured withglucose and acetate as the sole carbon source, respectively.Results showed that the growth rate of Wt/mdh and Ym/mdh was slightlyfaster than that of Wt and Ym, respectively, with glucose as the sole carbon,probably because more NADPH participating in the biosynthesis were produced byNADP-MDH (G³⁴S³⁵S³⁶G⁷⁷A⁷⁹), which promoted the growth of Wt/mdh andYm/mdh. However, growth rates of Wt/mdh and Ym/mdh declined remarkably, andwere just about28%and42%of Wt, respectively, with acetate as the sole carbonsource. These results implied that NAD-MDH was crucial for bacteria growth onthe barren carbon resource. Comparing the growth rates of the four strains onacetate, it was found that the influence on bacteria growth caused by coenzymespecificity of MDH was more evident than that of IDH. In addition, the promotingeffect on bacteria growth by the NADP-MDH (G³⁴S³⁵S³⁶G⁷⁷A⁷⁹) was not obviouson acetate, which mainly because the activity of engineered NADP-MDH wasrelatively low.