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Biochimica Et Biophysica Acta.... Nov 2019The crystal structure of the enzyme previously characterized as a type-2 NADH:menaquinone oxidoreductase (NDH-2) from Thermus thermophilus has been solved at a...
The crystal structure of the enzyme previously characterized as a type-2 NADH:menaquinone oxidoreductase (NDH-2) from Thermus thermophilus has been solved at a resolution of 2.9 Å and revealed that this protein is, in fact, a coenzyme A-disulfide reductase (CoADR). Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Thermus thermophilus and is maintained in the reduced state by this enzyme (CoADR). Although the enzyme does exhibit NADH:menadione oxidoreductase activity expected for NDH-2 enzymes, the specific activity with CoAD as an electron acceptor is about 5-fold higher than with menadione. Furthermore, the crystal structure contains coenzyme A covalently linked Cys44, a catalytic intermediate (Cys44-S-S-CoA) reduced by NADH via the FAD cofactor. Soaking the crystals with menadione shows that menadione can bind to a site near the redox active FAD, consistent with the observed NADH:menadione oxidoreductase activity. CoADRs from other species were also examined and shown to have measurable NADH:menadione oxidoreductase activity. Although a common feature of this family of enzymes, no biological relevance is proposed. The CoADR from T. thermophilus is a soluble homodimeric enzyme. Expression of the recombinant TtCoADR at high levels in E. coli results in a small fraction that co-purifies with the membrane fraction, which was used previously to isolate the enzyme wrongly identified as a membrane-bound NDH-2. It is concluded that T. thermophilus does not contain an authentic NDH-2 component in its aerobic respiratory chain.
Topics: Coenzyme A; Escherichia coli; Models, Molecular; NADH, NADPH Oxidoreductases; Recombinant Proteins; Static Electricity; Thermus thermophilus; Vitamin K 3; X-Ray Diffraction
PubMed: 31520616
DOI: 10.1016/j.bbabio.2019.148080 -
International Journal of Molecular... Oct 2023Fatty acid metabolism, including β-oxidation (βOX), plays an important role in human physiology and pathology. βOX is an essential process in the energy metabolism of... (Review)
Review
Fatty acid metabolism, including β-oxidation (βOX), plays an important role in human physiology and pathology. βOX is an essential process in the energy metabolism of most human cells. Moreover, βOX is also the source of acetyl-CoA, the substrate for (a) ketone bodies synthesis, (b) cholesterol synthesis, (c) phase II detoxication, (d) protein acetylation, and (d) the synthesis of many other compounds, including N-acetylglutamate-an important regulator of urea synthesis. This review describes the current knowledge on the importance of the mitochondrial and peroxisomal βOX in various organs, including the liver, heart, kidney, lung, gastrointestinal tract, peripheral white blood cells, and other cells. In addition, the diseases associated with a disturbance of fatty acid oxidation (FAO) in the liver, heart, kidney, lung, alimentary tract, and other organs or cells are presented. Special attention was paid to abnormalities of FAO in cancer cells and the diseases caused by mutations in gene-encoding enzymes involved in FAO. Finally, issues related to α- and ω- fatty acid oxidation are discussed.
Topics: Humans; Acyl Coenzyme A; Fatty Acids; Oxidation-Reduction; Liver; Acetyl Coenzyme A
PubMed: 37834305
DOI: 10.3390/ijms241914857 -
The Journal of Biological Chemistry May 2019ATP-citrate lyase (ACLY) is a major source of nucleocytosolic acetyl-CoA, a fundamental building block of carbon metabolism in eukaryotes. ACLY is aberrantly regulated...
ATP-citrate lyase (ACLY) is a major source of nucleocytosolic acetyl-CoA, a fundamental building block of carbon metabolism in eukaryotes. ACLY is aberrantly regulated in many cancers, cardiovascular disease, and metabolic disorders. However, the molecular mechanisms determining ACLY activity and function are unclear. To this end, we investigated the role of the uncharacterized ACLY C-terminal citrate synthase homology domain in the mechanism of acetyl-CoA formation. Using recombinant, purified ACLY and a suite of biochemical and biophysical approaches, including analytical ultracentrifugation, dynamic light scattering, and thermal stability assays, we demonstrated that the C terminus maintains ACLY tetramerization, a conserved and essential quaternary structure and likely also Furthermore, we show that the C terminus, only in the context of the full-length enzyme, is necessary for full ACLY binding to CoA. Together, we demonstrate that ACLY forms a homotetramer through the C terminus to facilitate CoA binding and acetyl-CoA production. Our findings highlight a novel and unique role of the C-terminal citrate synthase homology domain in ACLY function and catalysis, adding to the understanding of the molecular basis for acetyl-CoA synthesis by ACLY. This newly discovered means of ACLY regulation has implications for the development of novel ACLY modulators to target acetyl-CoA-dependent cellular processes for potential therapeutic use.
Topics: ATP Citrate (pro-S)-Lyase; Catalysis; Coenzyme A; Enzyme Stability; Protein Multimerization; Substrate Specificity; Temperature
PubMed: 30877197
DOI: 10.1074/jbc.RA118.006685 -
Biochemistry Mar 2013Linus Pauling proposed that the large rate accelerations for enzymes are caused by the high specificity of the protein catalyst for binding the reaction transition... (Review)
Review
Linus Pauling proposed that the large rate accelerations for enzymes are caused by the high specificity of the protein catalyst for binding the reaction transition state. The observation that stable analogues of the transition states for enzymatic reactions often act as tight-binding inhibitors provided early support for this simple and elegant proposal. We review experimental results that support the proposal that Pauling's model provides a satisfactory explanation for the rate accelerations for many heterolytic enzymatic reactions through high-energy reaction intermediates, such as proton transfer and decarboxylation. Specificity in transition state binding is obtained when the total intrinsic binding energy of the substrate is significantly larger than the binding energy observed at the Michaelis complex. The results of recent studies that aimed to characterize the specificity in binding of the enolate oxygen at the transition state for the 1,3-isomerization reaction catalyzed by ketosteroid isomerase are reviewed. Interactions between pig heart succinyl-coenzyme A:3-oxoacid coenzyme A transferase (SCOT) and the nonreacting portions of coenzyme A (CoA) are responsible for a rate increase of 3 × 10(12)-fold, which is close to the estimated total 5 × 10(13)-fold enzymatic rate acceleration. Studies that partition the interactions between SCOT and CoA into their contributing parts are reviewed. Interactions of the protein with the substrate phosphodianion group provide an ~12 kcal/mol stabilization of the transition state for the reactions catalyzed by triosephosphate isomerase, orotidine 5'-monophosphate decarboxylase, and α-glycerol phosphate dehydrogenase. The interactions of these enzymes with the substrate piece phosphite dianion provide a 6-8 kcal/mol stabilization of the transition state for reaction of the appropriate truncated substrate. Enzyme activation by phosphite dianion reflects the higher dianion affinity for binding to the enzyme-transition state complex compared with that of the free enzyme. Evidence is presented that supports a model in which the binding energy of the phosphite dianion piece, or the phosphodianion group of the whole substrate, is utilized to drive an enzyme conformational change from an inactive open form E(O) to an active closed form E(C), by closure of a phosphodianion gripper loop. Members of the enolase and haloalkanoic acid dehalogenase superfamilies use variable capping domains to interact with nonreacting portions of the substrate and sequester the substrate from interaction with bulk solvent. Interactions of this capping domain with the phenyl group of mandelate have been shown to activate mandelate racemase for catalysis of deprotonation of α-carbonyl carbon. We propose that an important function of these capping domains is to utilize the binding interactions with nonreacting portions of the substrate to activate the enzyme for catalysis.
Topics: Amino Acid Isomerases; Animals; Catalysis; Coenzyme A; Coenzyme A-Transferases; Enzyme Activation; Enzymes; Glycerolphosphate Dehydrogenase; Kinetics; Models, Chemical; Models, Molecular; Orotidine-5'-Phosphate Decarboxylase; Phosphopyruvate Hydratase; Substrate Specificity; Swine; Thermodynamics; Triose-Phosphate Isomerase
PubMed: 23327224
DOI: 10.1021/bi301491r -
Journal of Medicinal Chemistry Apr 2023Acute kidney injury (AKI) is associated with high morbidity and mortality, and no drugs are available clinically. Metabolic reprogramming resulting from the deletion of...
Acute kidney injury (AKI) is associated with high morbidity and mortality, and no drugs are available clinically. Metabolic reprogramming resulting from the deletion of S-nitroso-coenzyme A reductase 2 (SCoR2; AKR1A1) protects mice against AKI, identifying SCoR2 as a potential drug target. Of the few known inhibitors of SCoR2, none are selective versus the related oxidoreductase AKR1B1, limiting therapeutic utility. To identify SCoR2 (AKR1A1) inhibitors with selectivity versus AKR1B1, analogs of the nonselective (dual 1A1/1B1) inhibitor imirestat were designed, synthesized, and evaluated. Among 57 compounds, JSD26 has 10-fold selectivity for SCoR2 versus AKR1B1 and inhibits SCoR2 potently through an uncompetitive mechanism. When dosed orally to mice, JSD26 inhibited SNO-CoA metabolic activity in multiple organs. Notably, intraperitoneal injection of JSD26 in mice protected against AKI through S-nitrosylation of pyruvate kinase M2 (PKM2), whereas imirestat was not protective. Thus, selective inhibition of SCoR2 has therapeutic potential to treat acute kidney injury.
Topics: Mice; Animals; Oxidoreductases; Acute Kidney Injury; Coenzyme A; Kidney
PubMed: 37027003
DOI: 10.1021/acs.jmedchem.2c02089 -
Applied and Environmental Microbiology Feb 2022The acyl-coenzyme A (CoA) dehydrogenase family enzyme DmdC catalyzes the third step in the dimethylsulfoniopropionate (DMSP) demethylation pathway, the oxidation of...
The acyl-coenzyme A (CoA) dehydrogenase family enzyme DmdC catalyzes the third step in the dimethylsulfoniopropionate (DMSP) demethylation pathway, the oxidation of 3-methylmercaptopropionyl-CoA (MMPA-CoA) to 3-methylthioacryloyl-CoA (MTA-CoA). To study its substrate specificity, the recombinant DmdC1 from Ruegeria pomeroyi was characterized. In addition to MMPA-CoA, the enzyme was highly active with short-chain acyl-CoAs, with values for MMPA-CoA, butyryl-CoA, valeryl-CoA, caproyl-CoA, heptanoyl-CoA, caprylyl-CoA, and isobutyryl-CoA of 36, 19, 7, 11, 14, 10, and 149 μM, respectively, and values of 1.48, 0.40, 0.48, 0.73, 0.46, 0.23, and 0.01 s, respectively. Among these compounds, MMPA-CoA was the best substrate. The high affinity of DmdC1 for its substrate supports the model for kinetic regulation of the demethylation pathway. In contrast to DmdB, which catalyzes the formation of MMPA-CoA from MMPA, CoA, and ATP, DmdC1 was not affected by physiological concentrations of potential effectors, such as DMSP, MMPA, ATP, and ADP. Thus, compared to the other enzymes of the DMSP demethylation pathway, DmdC1 has only minimal adaptations for DMSP metabolism compared to other enzymes in the same family with similar substrates, supporting the hypothesis that it evolved relatively recently from a short-chain acyl-CoA dehydrogenase involved in fatty acid oxidation. We report the kinetic properties of DmdC1 from the model organism and close an important gap in the literature. While the crystal structure of this enzyme was recently solved and its mechanism of action described (X. Shao, H. Y. Cao, F. Zhao, M. Peng, et al., Mol Microbiol 111:1057-1073, 2019, https://doi.org/10.1111/mmi.14211), its substrate specificity and sensitivity to potential effectors was never examined. We show that DmdC1 has a high affinity for other short-chain acyl-CoAs in addition to MMPA-CoA, which is the natural substrate in DMSP metabolism and is not affected by the potential effectors tested. This evidence supports the hypothesis that DmdC1 possesses few adaptations to DMSP metabolism and likely evolved relatively recently from a short-chain acyl-CoA dehydrogenase involved in fatty acid oxidation. This work is important because it expands our understanding of the adaptation of marine bacteria to the increased availability of DMSP about 250 million years ago.
Topics: Bacterial Proteins; Coenzyme A; Oxidoreductases; Rhodobacteraceae; Substrate Specificity
PubMed: 34818101
DOI: 10.1128/AEM.01729-21 -
Applied and Environmental Microbiology Aug 2012Aromatic compounds (biogenic and anthropogenic) are abundant in the biosphere. Some of them are well-known environmental pollutants. Although the aromatic nucleus is... (Review)
Review
Aromatic compounds (biogenic and anthropogenic) are abundant in the biosphere. Some of them are well-known environmental pollutants. Although the aromatic nucleus is relatively recalcitrant, microorganisms have developed various catabolic routes that enable complete biodegradation of aromatic compounds. The adopted degradation pathways depend on the availability of oxygen. Under oxic conditions, microorganisms utilize oxygen as a cosubstrate to activate and cleave the aromatic ring. In contrast, under anoxic conditions, the aromatic compounds are transformed to coenzyme A (CoA) thioesters followed by energy-consuming reduction of the ring. Eventually, the dearomatized ring is opened via a hydrolytic mechanism. Recently, novel catabolic pathways for the aerobic degradation of aromatic compounds were elucidated that differ significantly from the established catabolic routes. The new pathways were investigated in detail for the aerobic bacterial degradation of benzoate and phenylacetate. In both cases, the pathway is initiated by transforming the substrate to a CoA thioester and all the intermediates are bound by CoA. The subsequent reactions involve epoxidation of the aromatic ring followed by hydrolytic ring cleavage. Here we discuss the novel pathways, with a particular focus on their unique features and occurrence as well as ecological significance.
Topics: Biodegradation, Environmental; Coenzyme A; Epoxy Compounds; Esters; Hydrocarbons, Aromatic; Hydrolysis; Metabolic Networks and Pathways; Molecular Structure; Oxygen
PubMed: 22582071
DOI: 10.1128/AEM.00633-12 -
Metabolic Engineering Nov 2017Malonyl-CoA is the basic building block for synthesizing a range of important compounds including fatty acids, phenylpropanoids, flavonoids and non-ribosomal... (Review)
Review
Malonyl-CoA is the basic building block for synthesizing a range of important compounds including fatty acids, phenylpropanoids, flavonoids and non-ribosomal polyketides. Centering around malonyl-CoA, we summarized here the various metabolic engineering strategies employed recently to regulate and control malonyl-CoA metabolism and improve cellular productivity. Effective metabolic engineering of microorganisms requires the introduction of heterologous pathways and dynamically rerouting metabolic flux towards products of interest. Transcriptional factor-based biosensors translate an internal cellular signal to a transcriptional output and drive the expression of the designed genetic/biomolecular circuits to compensate the activity loss of the engineered biosystem. Recent development of genetically-encoded malonyl-CoA sensor has stood out as a classical example to dynamically reprogram cell metabolism for various biotechnological applications. Here, we reviewed the design principles of constructing a transcriptional factor-based malonyl-CoA sensor with superior detection limit, high sensitivity and broad dynamic range. We discussed various synthetic biology strategies to remove pathway bottleneck and how genetically-encoded metabolite sensor could be deployed to improve pathway efficiency. Particularly, we emphasized that integration of malonyl-CoA sensing capability with biocatalytic function would be critical to engineer efficient microbial cell factory. Biosensors have also advanced beyond its classical function of a sensor actuator for in situ monitoring of intracellular metabolite concentration. Applications of malonyl-CoA biosensors as a sensor-invertor for negative feedback regulation of metabolic flux, a metabolic switch for oscillatory balancing of malonyl-CoA sink pathway and source pathway and a screening tool for engineering more efficient biocatalyst are also presented in this review. We envision the genetically-encoded malonyl-CoA sensor will be an indispensable tool to optimize cell metabolism and cost-competitively manufacture malonyl-CoA-derived compounds.
Topics: Biosensing Techniques; Malonyl Coenzyme A; Metabolic Engineering; Microorganisms, Genetically-Modified
PubMed: 29097310
DOI: 10.1016/j.ymben.2017.10.011 -
Applied and Environmental Microbiology Jul 2020Anaerobic degradation of polycyclic aromatic hydrocarbons has been investigated mostly with naphthalene as a model compound. Naphthalene degradation by sulfate-reducing...
Anaerobic degradation of polycyclic aromatic hydrocarbons has been investigated mostly with naphthalene as a model compound. Naphthalene degradation by sulfate-reducing bacteria proceeds via carboxylation to 2-naphthoic acid, formation of a coenzyme A thioester, and subsequent reduction to 5,6,7,8-tetrahydro-2-naphthoyl-coenzyme A (THNCoA), which is further reduced to hexahydro-2-naphthoyl-CoA (HHNCoA) by tetrahydronaphthoyl-CoA reductase (THNCoA reductase), an enzyme similar to class I benzoyl-CoA reductases. When analyzing THNCoA reductase assays with crude cell extracts and NADH as electron donor via liquid chromatography-mass spectrometry (LC-MS), scanning for putative metabolites, we found that small amounts of the product of an HHNCoA hydratase were formed in the assays, but the downstream conversion by an NAD-dependent β-hydroxyacyl-CoA dehydrogenase was prevented by the excess of NADH in those assays. Experiments with alternative electron donors indicated that 2-oxoglutarate can serve as an indirect electron donor for the THNCoA-reducing system via a 2-oxoglutarate:ferredoxin oxidoreductase. With 2-oxoglutarate as electron donor, THNCoA was completely converted and further metabolites resulting from subsequent β-oxidation-like reactions and hydrolytic ring cleavage were detected. These metabolites indicate a downstream pathway with water addition to HHNCoA and ring fission via a hydrolase acting on a β'-hydroxy-β-oxo-decahydro-2-naphthoyl-CoA intermediate. Formation of the downstream intermediate -2-carboxycyclohexylacetyl-CoA, which is the substrate for the previously described lower degradation pathway leading to the central metabolism, completes the anaerobic degradation pathway of naphthalene. Anaerobic degradation of polycyclic aromatic hydrocarbons is poorly investigated despite its significance in anoxic sediments. Using alternative electron donors for the 5,6,7,8-tetrahydro-2-naphthoyl-CoA reductase reaction, we observed intermediary metabolites of anaerobic naphthalene degradation via enzyme assays with cell extracts of anaerobic naphthalene degraders. The identified metabolites provide evidence that ring reduction terminates at the stage of hexahydro-2-naphthoyl-CoA and a sequence of β-oxidation-like degradation reactions starts with a hydratase acting on this intermediate. The final product of this reaction sequence was identified as -2-carboxycyclohexylacetyl-CoA, a compound for which a further downstream degradation pathway has recently been published (P. Weyrauch, A. V. Zaytsev, S. Stephan, L. Kocks, et al., Environ Microbiol 19:2819-2830, 2017, https://doi.org/10.1111/1462-2920.13806). Our study reveals the first ring-cleaving reaction in the anaerobic naphthalene degradation pathway. It closes the gap between the reduction of the first ring of 2-naphthoyl-CoA by 2-napthoyl-CoA reductase and the lower degradation pathway starting from -2-carboxycyclohexylacetyl-CoA, where the second ring cleavage takes place.
Topics: Anaerobiosis; Bacterial Proteins; Coenzyme A; Deltaproteobacteria; Naphthalenes; Oxidation-Reduction; Oxidoreductases
PubMed: 32444470
DOI: 10.1128/AEM.00996-20 -
PLoS Biology Mar 2016Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are...
Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). Here, we report two high-resolution PduL crystal structures with bound substrates. The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen.
Topics: Amino Acid Sequence; Bacterial Structures; Catalytic Domain; Coenzyme A; Molecular Sequence Data; Phosphate Acetyltransferase; Protein Conformation; Salmonella enterica
PubMed: 26959993
DOI: 10.1371/journal.pbio.1002399