Dataset Information

Engineering a d-lactate dehydrogenase that can super-efficiently utilize NADPH and NADH as cofactors.

ABSTRACT: Engineering the cofactor specificity of a natural enzyme often results in a significant decrease in its activity on original cofactor. Here we report that a NADH-dependent dehydrogenase (d-LDH) from Lactobacillus delbrueckii 11842 can be rationally engineered to efficiently use both NADH and NADPH as cofactors. Point mutations on three amino acids (D176S, I177R, F178T) predicted by computational analysis resulted in a modified enzyme designated as d-LDH*. The Kcat/Km of the purified d-LDH* on NADPH increased approximately 184-fold while the Kcat/Km on NADH also significantly increased, showing for the first time that a rationally engineered d-LDH could exhibit comparable activity on both NADPH and NADH. Further kinetic analysis revealed that the enhanced affinity with NADH or NADPH and the significant increased Kcat of d-LDH* resulted in the significant increase of d-LDH* activity on both NADPH and NADH. This study thus demonstrated that the cofactor specificity of dehydrogenase can be broadened by using targeted engineering approach, and the engineered enzyme can efficiently function in NADH-rich, or NADPH-rich, or NADH and NADPH-rich environment.

SUBMITTER: Meng H

PROVIDER: S-EPMC4842997 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Engineering a d-lactate dehydrogenase that can super-efficiently utilize NADPH and NADH as cofactors.

Meng Hengkai H Liu Pi P Sun Hongbing H Cai Zhen Z Zhou Jie J Lin Jianping J Li Yin Y

Scientific reports 20160425

Engineering the cofactor specificity of a natural enzyme often results in a significant decrease in its activity on original cofactor. Here we report that a NADH-dependent dehydrogenase (d-LDH) from Lactobacillus delbrueckii 11842 can be rationally engineered to efficiently use both NADH and NADPH as cofactors. Point mutations on three amino acids (D176S, I177R, F178T) predicted by computational analysis resulted in a modified enzyme designated as d-LDH*. The Kcat/Km of the purified d-LDH* on NA ...[more]

PMID: 27109778

Similar Datasets

Project description:UnlabelledPrevious studies of the oral pathogen Streptococcus mutans have determined that this Gram-positive facultative anaerobe mounts robust responses to both acid and oxidative stresses. The water-forming NADH oxidase (Nox; encoded by nox) is thought to be critical for the regeneration of NAD(+), for use in glycolysis, and for the reduction of oxygen, thereby preventing the formation of damaging reactive oxygen species. In this study, the free NAD(+)/NADH ratio in a nox deletion strain (Δnox) was discovered to be remarkably higher than that in the parent strain, UA159, when the strains were grown in continuous culture. This unanticipated result was explained by significantly elevated lactate dehydrogenase (Ldh; encoded by ldh) activity and ldh transcription in the Δnox strain, which was mediated in part by the redox-sensing regulator Rex. cDNA microarray analysis of S. mutans cultures exposed to simultaneous acid stress (growth at a low pH) and oxidative stress (generated through the deletion of nox or the addition of exogenous oxygen) revealed a stress response synergistically heightened over that with either stress alone. In the Δnox strain, this elevated stress response included increased glucose phosphoenolpyruvate phosphotransferase system (PTS) activity, which appeared to be due to elevated manL transcription, mediated in part, like elevated ldh transcription, by Rex. While the Δnox strain does possess a membrane composition different from that of the parent strain, it did not appear to have defects in either membrane permeability or ATPase activity. However, the altered transcriptome and metabolome of the Δnox strain were sufficient to impair its ability to compete with commensal peroxigenic oral streptococci during growth under aerobic conditions.ImportanceStreptococcus mutans is an oral pathogen whose ability to outcompete commensal oral streptococci is strongly linked to the formation of dental caries. Previous work has demonstrated that the S. mutans water-forming NADH oxidase is critical for both carbon metabolism and the prevention of oxidative stress. The results of this study show that upregulation of lactate dehydrogenase, mediated through the redox sensor Rex, overcompensates for the loss of nox. Additionally, nox deletion led to the upregulation of mannose and glucose transport, also mediated through Rex. Importantly, the loss of nox rendered S. mutans defective in its ability to compete directly with two species of commensal streptococci, suggesting a role for nox in the pathogenic potential of this organism.

Project description:Bacterial membrane-associated NAD-independent d-lactate dehydrogenase (Fe-S d-iLDH) oxidizes d-lactate into pyruvate. A sequence analysis of the enzyme reveals that it contains an Fe-S oxidoreductase domain in addition to a flavin adenine dinucleotide (FAD)-containing dehydrogenase domain, which differs from other typical d-iLDHs. Fe-S d-iLDH from Pseudomonas putida KT2440 was purified as a His-tagged protein and characterized in detail. This monomeric enzyme exhibited activities with l-lactate and several d-2-hydroxyacids. Quinone was shown to be the preferred electron acceptor of the enzyme. The two domains of the enzyme were then heterologously expressed and purified separately. The Fe-S cluster-binding motifs predicted by sequence alignment were preliminarily verified by site-directed mutagenesis of the Fe-S oxidoreductase domain. The FAD-containing dehydrogenase domain retained 2-hydroxyacid-oxidizing activity, although it decreased compared to the full Fe-S d-iLDH. Compared to the intact enzyme, the FAD-containing dehydrogenase domain showed increased catalytic efficiency with cytochrome c as the electron acceptor, but it completely lost the ability to use coenzyme Q10 Additionally, the FAD-containing dehydrogenase domain was no longer associated with the cell membrane, and it could not support the utilization of d-lactate as a carbon source. Based on the results obtained, we conclude that the Fe-S oxidoreductase domain functions as an electron transfer component to facilitate the utilization of quinone as an electron acceptor by Fe-S d-iLDH, and it helps the enzyme associate with the cell membrane. These functions make the Fe-S oxidoreductase domain crucial for the in vivo d-lactate utilization function of Fe-S d-iLDH.IMPORTANCE Lactate metabolism plays versatile roles in most domains of life. Lactate utilization processes depend on certain enzymes to oxidize lactate to pyruvate. In recent years, novel bacterial lactate-oxidizing enzymes have been continually reported, including the unique NAD-independent d-lactate dehydrogenase that contains an Fe-S oxidoreductase domain besides the typical flavin-containing domain (Fe-S d-iLDH). Although Fe-S d-iLDH is widely distributed among bacterial species, the investigation of it is insufficient. Fe-S d-iLDH from Pseudomonas putida KT2440, which is the major d-lactate-oxidizing enzyme for the strain, might be a representative of this type of enzyme. A study of it will be helpful in understanding the detailed mechanisms underlying the lactate utilization processes.

Project description:Gluconobacter oxydans has the unique property of a glucose oxidation system in the periplasmic space, where glucose is oxidized incompletely to ketogluconic acids in a nicotinamide cofactor-independent manner. Elimination of the gdhM gene for membrane-bound glucose dehydrogenase, the first enzyme for the periplasmic glucose oxidation system, induces a metabolic change whereby glucose is oxidized in the cytoplasm to acetic acid. G. oxydans strain NBRC3293 possesses two molecular species of type II NADH dehydrogenase (NDH), the primary and auxiliary NDHs that oxidize NAD(P)H by reducing ubiquinone in the cell membrane. The substrate specificities of the two NDHs are different from each other: primary NDH (p-NDH) oxidizes NADH specifically but auxiliary NDH (a-NDH) oxidizes both NADH and NADPH. We constructed G. oxydans NBRC3293 derivatives defective in the ndhA gene for a-NDH, in the gdhM gene, and in both. Our ΔgdhM derivative yielded higher cell biomass on glucose, as reported previously, but grew at a lower rate than the wild-type strain. The ΔndhA derivative showed growth behavior on glucose similar to that of the wild type. The ΔgdhM ΔndhA double mutant showed greatly delayed growth on glucose, but its cell biomass was similar to that of the ΔgdhM strain. The double mutant accumulated intracellular levels of NAD(P)H and thus shifted the redox balance to reduction. Accumulated NAD(P)H levels might repress growth on glucose by limiting oxidative metabolisms in the cytoplasm. We suggest that a-NDH plays a crucial role in redox homeostasis of nicotinamide cofactors in the absence of the periplasmic oxidation system in G. oxydans IMPORTANCE Nicotinamide cofactors NAD+ and NADP+ mediate redox reactions in metabolism. Gluconobacter oxydans, a member of the acetic acid bacteria, oxidizes glucose incompletely in the periplasmic space-outside the cell. This incomplete oxidation of glucose is independent of nicotinamide cofactors. However, if the periplasmic oxidation of glucose is abolished, the cells oxidize glucose in the cytoplasm by reducing nicotinamide cofactors. Reduced forms of nicotinamide cofactors are reoxidized by NADH dehydrogenase (NDH) on the cell membrane. We found that two kinds of NDH in G. oxydans have different substrate specificities: the primary enzyme is NADH specific, and the auxiliary one oxidizes both NADH and NADPH. Inactivation of the latter enzyme in G. oxydans cells in which we had induced cytoplasmic glucose oxidation resulted in elevated intracellular levels of NAD(P)H, limiting cell growth on glucose. We suggest that the auxiliary enzyme is important if G. oxydans grows independently of the periplasmic oxidation system.

Dataset Information

Engineering a d-lactate dehydrogenase that can super-efficiently utilize NADPH and NADH as cofactors.

Publications

Engineering a d-lactate dehydrogenase that can super-efficiently utilize NADPH and NADH as cofactors.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets