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Methanoarchaeal sulfolactate dehydrogenase: prototype of a new family of NADH-dependent enzymes.

ABSTRACT: The crystal structure of the sulfolactate dehydrogenase from the hyperthermophilic and methanogenic archaeon Methanocaldococcus jannaschii was solved at 2.5 A resolution (PDB id. 1RFM). The asymmetric unit contains a tetramer of tight dimers. This structure, complexed with NADH, does not contain a cofactor-binding domain with 'Rossmann-fold' topology. Instead, the tertiary and quaternary structures indicate a novel fold. The NADH is bound in an extended conformation in each active site, in a manner that explains the pro-S specificity. Cofactor binding involves residues belonging to both subunits within the tight dimers, which are therefore the smallest enzymatically active units. The protein was found to be a homodimer in solution by size-exclusion chromatography, analytical ultracentrifugation and small-angle neutron scattering. Various compounds were tested as putative substrates. The results indicate the existence of a substrate discrimination mechanism, which involves electrostatic interactions. Based on sequence homology and phylogenetic analyses, several other enzymes were classified as belonging to this novel family of homologous (S)-2-hydroxyacid dehydrogenases.

SUBMITTER: Irimia A

PROVIDER: S-EPMC381418 | biostudies-literature | 2004 Mar

REPOSITORIES: biostudies-literature

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Methanoarchaeal sulfolactate dehydrogenase: prototype of a new family of NADH-dependent enzymes.

Irimia Adriana A Madern Dominique D Zaccaï Giuseppe G Vellieux Frédéric M D FM

The EMBO journal 20040311 6

The crystal structure of the sulfolactate dehydrogenase from the hyperthermophilic and methanogenic archaeon Methanocaldococcus jannaschii was solved at 2.5 A resolution (PDB id. 1RFM). The asymmetric unit contains a tetramer of tight dimers. This structure, complexed with NADH, does not contain a cofactor-binding domain with 'Rossmann-fold' topology. Instead, the tertiary and quaternary structures indicate a novel fold. The NADH is bound in an extended conformation in each active site, in a man ...[more]

PMID: 15014443

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Project description:Thorough study of composition and fluorescence properties of a commercial reagent of active equine NAD-dependent alcohol dehydrogenase expressed and purified from E. coli has been carried out. Several experimental methods: spectral- and time-resolved two-photon excited fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fast protein liquid chromatography, and mass spectrometry were used for analysis. The reagent under study was found to contain also a number of natural fluorophores: free NAD(P)H, NADH-alcohol dehydrogenase, NADPH-isocitrate dehydrogenase, and pyridoxal 5-phosphate-serine hydroxymethyltransferase complexes. The results obtained demonstrated the potential and limitations of popular optical methods as FLIM for separation of fluorescence signals from free and protein-bound forms of NADH, NADPH, and FAD that are essential coenzymes in redox reactions in all living cells. In particular, NADH-alcohol dehydrogenase and NADPH-isocitrate dehydrogenase complexes could not be optically separated in our experimental conditions although fast protein liquid chromatography and mass spectrometry analysis undoubtedly indicated the presence of both enzymes in the molecular sample used. Also, the results of fluorescence, fast protein liquid chromatography, and mass spectrometry analysis revealed a significant contribution of the enzyme-bound coenzyme pyridoxal 5-phosphate to the fluorescence signal that could be separated from enzyme-bound NADH by using bandpass filters, but could effectively mask contribution from enzyme-bound FAD because the fluorescence spectra of the species practically overlapped. It was shown that enzyme-bound pyridoxal 5-phosphate fluorescence can be separated from enzyme-bound NAD(P)H and FAD through analysis of short fluorescence decay times of about tens of picoseconds. However, this analysis was found to be effective only at relatively high number of peak photon counts in recorded fluorescence signals. The results obtained in this study can be used for interpretation of fluorescence signals from a mixture of enzyme-bound fluorophores and should be taken into consideration when determining the intracellular NADH/FAD ratio using FLIM.

Project description:The bacterial lifestyle is plastic, requiring transcriptional, translational, and metabolic tailoring for survival. These dynamic cellular processes are energy intensive; therefore, flexible energetics is requisite for adaptive plasticity. An intricate network of complementary and supplementary pathways exists in bacterial energy metabolism. There are two main entry points for electrons in the aerobic electron transport system, NADH dehydrogenase (NDH) and succinate dehydrogenase (SDH), receiving electrons from NADH and succinate, respectively. Aerobic bacterial phyla have a non-proton-pumping NADH dehydrogenase, which is often the primary dehydrogenase under aerobiosis. Here, we report adaptive changes supporting growth restoration in an Escherichia coli strain lacking the primary dehydrogenase. Growth optimization is achieved by reducing the activity of succinate dehydrogenase, and thus we demonstrate a physiological discord between proton-pumping NADH dehydrogenase and succinate dehydrogenase in supporting growth. Beyond the fundamental understanding of the bioenergetic network, identifying this compensatory feature provides impetus to rational antimicrobial combinations for targeting the non-proton-pumping dehydrogenase. IMPORTANCE Energy generation pathways are a potential avenue for the development of novel antibiotics. However, bacteria possess remarkable resilience due to the compensatory pathways, which presents a challenge in this direction. NADH, the primary reducing equivalent, can transfer electrons to two distinct types of NADH dehydrogenases. Type I NADH dehydrogenase is an enzyme complex comprising multiple subunits and can generate proton motive force (PMF). Type II NADH dehydrogenase does not pump protons but plays a crucial role in maintaining the turnover of NAD+. To study the adaptive rewiring of energy metabolism, we evolved an Escherichia coli mutant lacking type II NADH dehydrogenase. We discovered that by modifying the flux through the tricarboxylic acid (TCA) cycle, E. coli could mitigate the growth impairment observed in the absence of type II NADH dehydrogenase. This research provides valuable insights into the intricate mechanisms employed by bacteria to compensate for disruptions in energy metabolism.

Dataset Information

Methanoarchaeal sulfolactate dehydrogenase: prototype of a new family of NADH-dependent enzymes.

Publications

Methanoarchaeal sulfolactate dehydrogenase: prototype of a new family of NADH-dependent enzymes.

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