Impaired tricarboxylic acid cycle flux and mitochondrial aerobic respiration during isoproterenol induced myocardial ischemia is rescued by bilobalide

There is an urgent need to elucidate the pathogenesis of myocardial ischemia(MI)and potential drug treatments.Here,the anti-MI mechanism and material basis of Ginkgo biloba L.extract(GBE)were studied from the perspective of energy metabolism flux regulation.Metabolic flux analysis(MFA)was performed to investigate energy metabolism flux disorder and the regulatory nodes of GBE components in isoproterenol(ISO)-induced ischemia-like cardiomyocytes.It showed that[U-13 C]glucose derived m+2 isotopologues from the upstream tricarboxylic acid(TCA)cycle metabolites were markedly accumulated in ISO-injured cardiomyocytes,but the opposite was seen for the downstream metabolites,while their total cellular concentrations were increased.This indicates a blockage of carbon flow from glycolysis and enhanced anaplerosis from other carbon sources.A Seahorse test was used to screen for GBE components with regulatory effects on mitochondrial aerobic respiratory dysfunction.It showed that bilobalide protected against impaired mitochondrial aerobic respiration.MFA also showed that bilobalide significantly modulated the TCA cycle flux,reduced abnormal metabolite accumulation,and balanced the demand of different carbon sources.Western blotting and PCR analysis showed that bilobalide decreased the enhanced expression of key metabolic enzymes in injured cells.Bilobalide’s efficacy was verified by in vivo experiments in rats.This is the first report to show that bilobalide,the active ingredient of GBE,protects against MI by rescuing impaired TCA cycle flux.This provides a new mechanism and potential drug treatment for MI.It also shows the potential of MFA/Seahorse combination as a powerful strategy for pharmacological research on herbal medicine.

Key genes expression of reductive tricarboxylic acid cycle from deep-sea hydrothermal chemolithoautotrophic Caminibacter profundus in response to salinity, pH and O_2

CO2 fixation pathway of Caminibacter profundus, a chemolithoautotrophic e-Proteobacteria from deep-sea hydrothermal vent, was determined and characterized by genetic and enzymatic analyses. Gene expression of key enzymes for CO2 fixation in response to salinity, pH and O2 in Medium 829 were also investigated. The results demonstrate that C. profundus contained aclB, porA and oorA, the genes encoding key enzymes of reductive tricarboxylic acid （rTCA） cycle. However, genes fragments of cbbL and cbbMencoding key enzyme of Calvin cycle were not recovered. Key enzymatic activities of ATP citrate lyase （ACL）, pyruvate： ferredoxin oxidoreductase （POR） and 2-oxoglutarate： ferredoxin oxidoreductase （OOR） were also present in C. profun- dus. The combination of genetic and enzymatic analyses confirm that C. profundus adopted rTCA cycle for carbon assimilation. The results of aclB and oorA relative expressions of C. profundus demonstrate that the ranges of environmental factors for high genes expression were sea salt 3.0%-5.0% （optimum 3.0%）, pH 5.0-6.5（optimum pH 6.5）, anaerobic to microaerobic conditions （optimum 1.0% 02）. Gene expression pat- terns under different conditions show similar patterns with bacterial growth, revealing that key rTCA cycle genes provided molecular basis for bacterial growth and propagation. Our results suggest that C. profun- dus could regulate key genes of rTCA cycle for carbon assimilation and energy metabolism in response to environmental fluctuations in hydrothermal vent.

Arachidyl amido cholanoic acid improves liver glucose and lipid homeostasis in nonalcoholic steatohepatitis via AMPK and mTOR regulation

BACKGROUND Arachidyl amido cholanoic acid(Aramchol)is a potent downregulator of hepatic stearoyl-CoA desaturase 1(SCD1)protein expression that reduces liver triglycerides and fibrosis in animal models of steatohepatitis.In a phase IIb clinical trial in patients with nonalcoholic steatohepatitis(NASH),52 wk of treatment with Aramchol reduced blood levels of glycated hemoglobin A1c,an indicator of glycemic control.AIM To assess lipid and glucose metabolism in mouse hepatocytes and in a NASH mouse model[induced with a 0.1%methionine and choline deficient diet(0.1MCD)]after treatment with Aramchol.METHODS Isolated primary mouse hepatocytes were incubated with 20μmol/L Aramchol or vehicle for 48 h.Subsequently,analyses were performed including Western blot,proteomics by mass spectrometry,and fluxomic analysis with 13C-uniformly labeled glucose.For the in vivo part of the study,male C57BL/6J mice were randomly fed a control or 0.1MCD for 4 wk and received 1 or 5 mg/kg/d Aramchol or vehicle by intragastric gavage for the last 2 wk.Liver metabolomics were assessed using ultra-high-performance liquid chromatography-time of flight-MS for the determination of glucose metabolism-related metabolites.RESULTS Combination of proteomics and Western blot analyses showed increased AMPK activity while the activity of nutrient sensor mTORC1 was decreased by Aramchol in hepatocytes.This translated into changes in the content of their downstream targets including proteins involved in fatty acid(FA)synthesis and oxidation[PACCα/β(S79),SCD1,CPT1A/B,HADHA,and HADHB],oxidative phosphorylation(NDUFA9,NDUFB11,NDUFS1,NDUFV1,ETFDH,and UQCRC2),tricarboxylic acid(TCA)cycle(MDH2,SUCLA2,and SUCLG2),and ribosome(P-p70S6K[T389]and P-S6[S235/S236]).Flux experiments with 13Cuniformely labeled glucose showed that TCA cycle cataplerosis was reduced by Aramchol in hepatocytes,as indicated by the increase in the number of rounds that malate remained in the TCA cycle.Finally,liver metabolomic analysis showed that glucose homeostasis was improved by Aramchol in 0.1MCD fed mice in a dose-dependent manner,showing normalization of glucose,G6P,F6P,UDP-glucose,and Rbl5P/Xyl5P.CONCLUSION Aramchol exerts its effect on glucose and lipid metabolism in NASH through activation of AMPK and inhibition of mTORC1,which in turn activate FAβ-oxidation and oxidative phosphorylation.

Effects of GA3 and ABA on the respiratory pathways during the secondary bud burst in black currants

The effects of exogenous gibberellic acid (GA(3)) and abscisic acid (ABA) on the total respiratory rate, percentages of total respiratory rate contributed by respiratory pathways [Embden-Meyerhof- Parnas Pathway (EMP), Pentose Phosphate Pathway (PPP), and Tricarboxylic Acid Cycle (TCA)], and conversion of starch to soluble sugars in the buds of black currants during secondary bud burst were investigated to determine the relationship between respiratory rates and secondary bud burst. 'Adelinia', a black currant cultivar that is prone to secondary bud burst after the first harvest, was used in this study. Mature bushes of Adelinia were sprayed with 30 mg/L GA(3) and 50 mg/L ABA to manipulate bud burst. The results showed that exogenous applications of GA(3) and ABA had opposite effects on bud respiratory rate. Generally, GA(3) treatment increased the total respiratory rate and respiratory rate of the TCA and PPP, and the respiratory rates after GA(3) treatment were higher than those of control. While ABA treatment mostly decreased the total respiratory rate and the respiratory rate of TCA and PPP in buds in comparison to control. In terms of the percentage of the three respiratory rates in comparison to the total respiratory rate, GA(3) treatment significantly increased the percentage of TCA and PPP respiratory rate in comparison to the control (P < 0.01), whereas ABA decreased the rates. GA(3) significantly increased the content of soluble sugars and decreased the starch content, while the starch content in buds after ABA treatment was significantly higher than that of the control. All results showed that PPP is a critical process for the second bud burst in black currants. While the EMP-TCA pathway is still dominant in bud respiration, provides a series of basic materials and energy (ATP). The conversion of starch to soluble sugars is essential for bud burst. Thus, we conclude that an energy shortage is a main contributor in ABA inhibition of the secondary bud burst of black currants.

Identification of Potential Therapeutic Targets of Alzheimer's Disease By Weighted Gene Co-Expression Network Analysis

Objective Alzheimer's disease(AD)is the most common cause of dementia.The pathophysiology of the disease mostly remains unearthed,thereby challenging drug development for AD.This study aims to screen high throughput gene expression data using weighted co-expression network analysis(WGCNA)to explore the potential therapeutic targets.Methods The dataset of GSE36980 was obtained from the Gene Expression Omnibus(GEO)database.Normalization,quality control,filtration,and soft-threshold calculation were carried out before clustering the co-expressed genes into different modules.Furthermore,the correlation coefiidents between the modules and clinical traits were computed to identify the key modules.Gene ontology and pathway enrichment analyses were performed on the key module genes.The STRING database was used to construct the protein-protein interaction(PPI)networks,which were further analyzed by Cytoscape app(MCODE).Finally,validation of hub genes was conducted by external GEO datasets of GSE 1297 and GSE 28146.Results Co-expressed genes were clustered into 27 modules,among which 6 modules were identified as the key module relating to AD occurrence.These key modules are primarily involved in chemical synaptic transmission(G0:0007268),the tricarboxylic acid(TCA)cycle and respiratory electron transport(R-HSA-1428517).WDR47,OXCT1,C3orfl4,ATP6V1A,SLC25A14,NAPB were found as the hub genes and their expression were validated by external datasets.Conclusions Through modules co-expression network analyses and PPI network analyses,we identified the hub genes of AD,including WDR47,0XCT1,C3orfl4i ATP6V1A,SLC25A14 and NAPB.Among them,three hub genes(ATP6V1A,SLC25A14,OXCT1)might contribute to AD pathogenesis through pathway of TCA cycle.

Cellular metabolic energy modulation by tangeretin in 7,12-dimethylbenz(a) anthracene-induced breast cancer

Breast cancer is the leading cause of death among women worldwide.Chemoprevention and chemotherapy play beneficial roles in reducing the incidence and mortality of cancer.Epidemiological and experimental studies showed that naturally-occurring antioxidants present in the diet may act as anticancer agents.Identifying the abnormalities of cellular energy metabolism facilitates early detection and management of breast cancer.The present study evaluated the effect of tangeretin on cellular metabolic energy fluxes in 7,12-dimethylbenz（a）anthracene（DMBA）-induced proliferative breast cancer.The results showed that the activities of glycolytic enzymes significantly increased in mammary tissues of DMBA-induced breast cancer bearing rats.The gluconeogenic tricarboxylic acid（TCA） cycle and respiratory chain enzyme activities significantly decreased in breast cancer-bearing rats.In addition,proliferating cell nuclear antigen（PCNA） was highly expressed in breast cancer tissues.However,the activities of glycolytic enzymes were significantly normalized in the tangeretin pre- and post-treated rats and the TCA cycle and respiratory chain enzyme activities were significantly increased in tangeretin treated rats.Furthermore,tangeretin down-regulated PCNA expression on breast cancerbearing rats.Our study demonstrates that tangeretin specifically regulates cellular metabolic energy fluxes in DMBA-induced breast cancer-bearing rats.

The LncRNA FEZF1-AS1 promotes tumor proliferation in colon cancer by regulating the mitochondrial protein PCK2

LncRNAs and metabolism represents two factors involved in cancer initiation and progression.However,the interaction between lncRNAs and metabolism remains to be fully explored.In this study,lncRNA FEZF1-AS1(FEZF1-AS1)was found upregulated in colon cancer after screening all the lncRNAs of colon cancer tissues deposited in TCGA,the result of which was further confirmed by RNAscope staining on a colon tissue chip.The results obtained using FEZF1-AS1 knockout colon cancer cells(SW480 KO and HCT-116 KO)constructed using CRISPR/Cas9 system confirmed the proliferation,invasion,and migration-promoting function of FEZF1-AS1 in vitro.Mechanistically,FEZF1-AS1 associated with the mitochondrial protein phosphoenolpyruvate carboxykinase(PCK2),which plays an essential role in regulating energy metabolism in the mitochondria.Knockdown of FEZF1-AS1 greatly decreased PCK2 protein levels,broke the homeostasis of energy metabolism in the mitochondria,and inhibited proliferation,invasion,and migration of SW480 and HCT-116 cells.PCK2 overexpression in FEZF1-AS1 knockout cells partially rescued the tumor inhibitory effect on colon cancer cells both in vitro and in vivo.Moreover,PCK2 overexpression specifically rescued the abnormal accumulation of Flavin mononucleotide(FMN)and succinate,both of which play an important role in oxidative phosphorylation(OXPHOS).Overall,these results indicate that FEZF1-AS1 is an oncogene through regulating energy metabolism of the cell.This research reveals a new mechanism for lncRNAs to regulate colon cancer and provides a potential target for colon cancer diagnosis and treatment.

Impact of post-ruminally infused macronutrients on bovine mammary gland expression of genes involved in fatty acid synthesis, energy metabolism, and protein synthesis measured in RNA isolated from milk fat

Background: Characterising the regulation of milk component synthesis in response to macronutrient supply is critical for understanding the implications of nutritional interventions on milk production. Gene expression in mammary gland secretory cells was measured using RNA isolated from milk fat globules from 6 Holstein-Friesian cows receiving 5-d abomasal infusions of saline, essential amino acids(AA), or glucose(GG) or palm olein(LG)without(LAA) or with(HAA) essential AA, according to a 6 × 6 Latin square design. RNA was isolated from milk fat samples collected on d 5 of infusion and subjected to real-time quantitative PCR. We hypothesised that m RNA expression of genes involved in de novo milk fatty acid(FA) synthesis would be differently affected by GG and LG,and that expression of genes regulating transfer of tricarboxylic acid cycle intermediates would increase at the HAA level. We also hypothesised that the HAA level would affect genes regulating endoplasmic reticulum(ER)homeostasis but would not affect genes related to the mechanistic target of rapamycin complex 1(m TORC1) or the integrated stress response(ISR) network.Results: Infusion of GG did not affect de novo milk FA yield but decreased expression of FA synthase(FASN).Infusion of LG decreased de novo FA yield and tended to decrease expression of acetyl-Co A carboxylase 1(ACC1).The HAA level increased both de novo FA yield and expression of ACC1, and tended to decrease expression of mitochondrial phosphoenolpyruvate carboxykinase(PCK2). m RNA expression of m TORC1 signaling participants was not affected by GG, LG, or AA level. Expression of the ε subunit of the ISR constituent eukaryotic translation initiation factor 2 B(EIF2 B5) tended to increase at the HAA level, but only in the presence of LG. X-box binding protein 1(XBP1) m RNA was activated in response to LG and the HAA level.Conclusions: Results show that expression of genes involved in de novo FA synthesis responded to glucogenic,lipogenic, and aminogenic substrates, whereas genes regulating intermediate flux through the tricarboxylic acid cycle were not majorly affected. Results also suggest that after 5 d of AA supplementation, milk protein synthesis is supported by enhanced ER biogenesis instead of signaling through the mTORC1 or ISR networks.

Fluorescence Quenching Method for Determination of Cu^(2+) with Tb-TMA System in Presence of Triton X-100

A fluorescence quenching system of Cu 2+ Tb TMA Triton X 100 (TMA: benzene 1,3,5 tricarboxylic acid) was established for the determination of Cu 2+ . The fluorescence intensity of the determination system is linear in the concentration range of 1.58×10 -7 ～2.83×10 -5 mol·L -1 Cu 2+ . The proposed method was applied to the determination of Cu 2+ contained in various copper ores with satisfactory results.

Chiral phenethylamine synergistic tricarboxylic acid modified β-cyclodextrin immobilized on porous silica for enantioseparation

In this work, a series of chiral phenethylamine synergistic tricarboxylic acid modified β-cyclodextrin bonded stationary phase for high performance liquid chromatography(HPLC) were synthesized via a simple one-pot synthesis approach. Various racemates(aryl alcohols, flavanones, triazoles, benzoin, etc.) were well separated on the tricarboxylic acid modified chiral stationary phases in both normal and reversed modes with good reproducibility and stability, and the influence of mobile phase composition on resolution(R_(s)) were deeply investigated. The RSD values of Rsfor repeatability and column-to-column were below 1.28% and 3.05%, respectively. Hence, the fabrication of tricarboxylic acid modified chiral stationary phase(CSPs) is a new efficient strategy to improve the application of β-cyclodextrin as CSPs in the field of chromatography.

On the role of the tricarboxylic acid cycle in plant productivity

The tricarboxylic acid(TCA) cycle is one of the canonical energy pathways of living systems, as well as being an example of a pathway in which dynamic enzyme assemblies, or metabolons, are well characterized. The role of the enzymes have been the subject of saturated transgenesis approaches, whereby the expression of the constituent enzymes were reduced or knocked out in order to ascertain their in vivo function.Some of the resultant plants exhibited improved photosynthesis and plant growth, under controlled greenhouse conditions. In addition, overexpression of the endogenous genes, or heterologous forms of a number of the enzymes, has been carried out in tomato fruit and the roots of a range of species, and in some instances improvement in fruit yield and postharvest properties and plant performance, under nutrient limitation, have been reported, respectively. Given a number of variants, in nature, we discuss possible synthetic approaches involving introducing these variants, or at least a subset of them, into plants. We additionally discuss the likely consequences of introducing synthetic metabolons, wherein certain pairs of reactions are artificially permanently assembled into plants, and speculate as to future strategies to further improve plant productivity by manipulation of the core metabolic pathway.

Detailed profiling of carbon fixation of in silico synthetic autotrophy with reductive tricarboxylic acid cycle and Calvin-Benson-Bassham cycle in Esherichia coli using hydrogen as an energy source

Carbon fixation is the main route of inorganic carbon in the form of CO2 into the biosphere.In nature,RuBisCO is the most abundant protein that photosynthetic organisms use to fix CO2 from the atmosphere through the Calvin-Benson-Bassham(CBB)cycle.However,the CBB cycle is limited by its low catalytic rate and low energy efficiency.In this work,we attempt to integrate the reductive tricarboxylic acid and CBB cycles in silico to further improve carbon fixation capacity.Key heterologous enzymes,mostly carboxylating enzymes,are inserted into the Esherichia coli core metabolic network to assimilate CO2 into biomass using hydrogen as energy source.Overall,such a strain shows enhanced growth yield with simultaneous running of dual carbon fixation cycles.Our key results include the following.(i)We identified two main growth states:carbon-limited and hydrogenlimited;(ii)we identified a hierarchy of carbon fixation usage when hydrogen supply is limited;and(iii)we identified the alternative sub-optimal growth mode while performing genetic perturbation.The results and modeling approach can guide bioengineering projects toward optimal production using such a strain as a microbial cell factory.

Metabolic dysregulation and emerging therapeutical targets for hepatocellular carcinoma

Hepatocellular carcinoma(HCC)is an aggressive human cancer with increasing incidence worldwide.Multiple efforts have been made to explore pharmaceutical therapies to treat HCC,such as targeted tyrosine kinase inhibitors,immune based therapies and combination of chemotherapy.However,limitations exist in current strategies including chemoresistance for instance.Tumor initiation and progression is driven by reprogramming of metabolism,in particular during HCC development.Recently,metabolic associated fatty liver disease(MAFLD),a reappraisal of new nomenclature for nonalcoholic fatty liver disease(NAFLD),indicates growing appreciation of metabolism in the pathogenesis of liver disease,including HCC,thereby suggesting new strategies by targeting abnormal metabolism for HCC treatment.In this review,we introduce directions by highlighting the metabolic targets in glucose,fatty acid,amino acid and glutamine metabolism,which are suitable for HCC pharmaceutical intervention.We also summarize and discuss current pharmaceutical agents and studies targeting deregulated metabolism during HCC treatment.Furthermore,opportunities and challenges in the discovery and development of HCC therapy targeting metabolism are discussed.

Application of ~1H NMR-based metabolomics for discrimination of different parts and development of a new processing workflow for Cistanche deserticola

Cistanche deserticola(CD) is one of the two authoritative source plants of Cistanches Herba, a well-known medicinal plant. Herein,~1H NMR spectroscopy was employed to characterize the chemical profile and to distinguish the different parts, as well as to propose a new processing workflow for CD.Signal assignment was achieved by multiple one and two dimensional NMR spectroscopic techniques in combination with available databases and authentic compounds. The upper parts of the plant were distinguished from the lower parts by combining ~1H NMR spectroscopic dataset with multivariate statistical analysis. A new processing method that hyphenated steaming with freeze-drying, was demonstrated to be superior to either steaming coupled with oven-drying or direct freeze-drying via holistic ~1H NMR-based metabolomic characterization. Phenylethanoid glycosides, mainly echinacoside and acteoside, were screened out and confirmed as the chemical markers responsible for exhibiting the superiority of the new processing workflow, whereas serial primary metabolites, especially carbohydrates and tricarboxylic acid cycle metabolites, were found as the primary molecules governing the discrimination between the upper and lower parts of the plant. Collectively,~1H NMR spectroscopy was demonstrated as a versatile analytical tool to characterize the chemical profile and to guide the indepth exploitation of CD by providing comprehensive qualitative and quantitative information.

Expression of EGFP/SDCT1 fusion protein,subcellular localization signal analysis,tissue distribution and electrophysiological function study

Full-length cDNA gene of sodium-dependent dicarboxylate co-transporter protein 1 (SDCT1) is cloned from normal human kidney tissue and inserted into EGFP (enhanced green fluorescent protein) expression vector along with N-terminal and C-terminal truncated SDCT1 genes, so to construct the eukaryotic expression vectors of EGFP/SDCT1 fusion proteins, which are transfected into human renal tubular epithelial cells (HKC). Subcellular localizations of these fusion proteins are observed by laser confocal microscope to determine the localization signal of the SDCT1 protein. Duplex PCR analysis validates that the fusion protein genes have been in- tegrated into the genome of HKC. Western blot indicates that the fusion proteins have been ex- pressed in HKC. Confocal microscopy analysis shows that human SDCT1 predominantly locates on the plasma membrane, which is consistent with the results predicted by bioinformatics ap- proach; in HKC transfected with N-terminal truncated SDCT1 gene, the green fluorescence is mainly distributed on the plasma membrane; in HKC transfected with C-terminal truncated SDCT1 gene, the green fluorescence is mainly distributed in the cytoplasm. EGFP/SDCT1 mRNAs obtained by in vitro transcription are microinjected into Xenopus laevis oocytes for ex- pression and the trans-membrane currents are measured by using two-microelectrode volt- age-clamp technique. Na+ inward currents are present on cellular membrane of the injected oo- cytes. Immunohistochemical staining shows that human SDCT1 proteins are expressed on lu- men membrane of the renal proximal tubule, but are negative in distal tubule, collecting duct, renal interstitium and glomerulus. The above-mentioned studies suggest that human SDCT1 protein is located on the lumen membrane of the renal proximal tubule, the C-terminal sequence of the SDCT1 is required for delivery and targeting localization, and the plasma membrane lo- calization signal of the SDCT1 protein maybe locate in the C-terminal sequence.

Syntheses,Structures and Catalytic Properties of Two Mn(Ⅱ)and Cd(Ⅱ)Coordination Polymers through in Situ Ligand Reaction

Two coordination polymers,namely[Mn(μ-Hcpia)(bipy)(H_(2)O)_(2)]_(n)(1)and[Cd_(3)(μ3-Hcpia)_(2)(μ-Hbiim)_(2)(μ-H_(2)biim)(H_(2)O)_(2)]n(2),have been constructed hydrothermally using H_(2)cbia(H_(2)cbia=5-(4΄-cyano-benzoxy)isophthalic acid),bipy(bipy=4,4΄-bipyridine),H_(2)biim(H_(2)biim=2,2΄-biimidazole),and manganese or cadmium chlorides at 160℃.Interestingly,the H3cpia(H3cpia=5-(4΄-carboxylphenoxy)isophthalic acid)ligand was generated by in situ hydrolysis of cyano group in H_(2)cbia.The products were isolated as stable crystalline solids and were characterized by IR spectra,elemental analyses,thermogravimetric analyses(TGA),and single-crystal X-ray diffraction analyses.Both compounds crystallize in the triclinic system,space group P1.Compound 1 discloses a 1D linear chain of the 2C1 topological type.Adjacent chains are assembled into a 2D supramolecular sheet through O–H∙∙∙O/N hydrogen bonds.Compound 2 features a 3D framework with a 3,4,4T25 topology.The luminescent and catalytic properties of two compounds were investigated.Compound 1 exhibits a superior catalytic activity in the cyanosilylation at room temperature.

Roles of Thioredoxins in the Obligate Anaerobic Green Sulfur Photosynthetic Bacterium Chlorobaculum tepidum

Thioredoxin is a small ubiquitous protein that is involved in the dithiol-disulfide exchange reaction, byway of two cysteine residues located on the molecule surface. In order to elucidate the role of thioredoxin in Chlorobaculum tepidurn, an anaerobic green sulfur bacterium that uses various inorganic sulfur compounds and H2S as electron donors under strict anaerobic conditions for growth, we applied the thioredoxin affinity chromatography method （Motohashi et al., 2001）. In this study, 37 cytoplasmic proteins were captured as thioredoxin target candidates, including proteins involved in sulfur assimilation. Furthermore, six of the candidate proteins were members of the reductive tricarboxylic acid cycle （pyruvate orthophosphate dikinase, pyruvate flavodoxin/ferredoxin oxidoreductase, ^-oxoglutarate synthase, citrate lyase, citrate synthase, malate dehydrogenase）. The redox sensitivity of three enzymes was then examined： citrate lyase, citrate synthase, and malate dehydrogenase, using their recombinant proteins. Based on the information relating to the target proteins, the significance of thioredoxin as a reductant for the metabolic pathway in the anaerobic photosynthetic bacteria is discussed.

Function, kinetic properties, crystallization, and regulation of microbial malate dehydrogenase

Malate dehydrogenase （MDH） is an enzyme widely distributed among living organisms and is a key protein in the central oxidative pathway. It catalyzes the interconversion between malate and oxaloacetate using NAD＋ or NADP＊ as a cofactor. Surprisingly, this enzyme has been extensively studied in eukaryotes but there are few reports about this enzyme in prokaryotes. It is necessary to review the relevant information to gain a better understanding of the function of this enzyme. Our review of the data generated from studies in bacteria shows much diversity in their molecular properties, including weight, oligomeric states, cofactor and substrate binding affinities, as well as differences in the direction of the enzymatic reaction. Furthermore, due to the importance of its function, the transcription and activity of this enzyme are rigorously regulated. Crystal structures of MDH from different bacterial sources led to the identification of the regions involved in substrate and cofactor binding and the residues important for the dimer-dimer interface. This structural information allows one to make direct modifications to improve the enzyme catalysis by increasing its activity, cofactor binding capacity, substrate specificity, and thermostability. A comparative analysis of the phylogenetic reconstruction of MDH reveals interesting facts about its evolutionary history, dividing this superfamily of proteins into two principle clades and establishing relationships between MDHs from different cellular compartments from archaea, bacteria, and eukaryotes.