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1.  Gut bacteria that rescue growth impairments transmitted by immature microbiota from undernourished children 
Science (New York, N.Y.)  2016;351(6275):10.1126/science.aad3311 aad3311.
Undernourished children exhibit impaired gut microbiota development. Transplanting microbiota from 6- and 18-month old healthy or undernourished Malawian donors into young germ-free mice fed a Malawian diet revealed that immature microbiota from undernourished infants/children transmit impaired growth phenotypes. The representation of several age-discriminatory taxa in recipient animals correlated with lean body mass gain, liver, muscle, and brain metabolism, plus bone morphology. Co-housing mice shortly after receiving microbiota from healthy (H) or severely stunted/underweight (Un) infants demonstrated that invasion of age-/growth-discriminatory taxa from H to Un cagemates’ microbiota ameliorates growth faltering. Adding two invasive species, Ruminococcus gnavus and Clostridium symbiosum, to the Un microbiota also ameliorated growth and metabolic abnormalities. These results provide evidence that microbiota immaturity is causally related to undernutrition, and reveal potential therapeutic targets and agents.
doi:10.1126/science.aad3311
PMCID: PMC4787260  PMID: 26912898
2.  Glutamate and asparagine cataplerosis underlie glutamine addiction in melanoma 
Oncotarget  2015;6(10):7379-7389.
Glutamine dependence is a prominent feature of cancer metabolism, and here we show that melanoma cells, irrespective of their oncogenic background, depend on glutamine for growth. A quantitative audit of how carbon from glutamine is used showed that TCA-cycle-derived glutamate is, in most melanoma cells, the major glutamine-derived cataplerotic output and product of glutaminolysis. In the absence of glutamine, TCA cycle metabolites were liable to depletion through aminotransferase-mediated α-ketoglutarate-to-glutamate conversion and glutamate secretion. Aspartate was an essential cataplerotic output, as melanoma cells demonstrated a limited capacity to salvage external aspartate. Also, the absence of asparagine increased the glutamine requirement, pointing to vulnerability in the aspartate-asparagine biosynthetic pathway within melanoma metabolism. In contrast to melanoma cells, melanocytes could grow in the absence of glutamine. Melanocytes use more glutamine for protein synthesis rather than secreting it as glutamate and are less prone to loss of glutamate and TCA cycle metabolites when starved of glutamine.
PMCID: PMC4480687  PMID: 25749035
Melanoma; Glutaminolysis; Cataplerosis; Stable isotopes
3.  Metabolic and Bactericidal Effects of Targeted Suppression of NadD and NadE Enzymes in Mycobacteria 
mBio  2014;5(1):e00747-13.
ABSTRACT
Mycobacterium tuberculosis remains a major cause of death due to the lack of treatment accessibility, HIV coinfection, and drug resistance. Development of new drugs targeting previously unexplored pathways is essential to shorten treatment time and eliminate persistent M. tuberculosis. A promising biochemical pathway which may be targeted to kill both replicating and nonreplicating M. tuberculosis is the biosynthesis of NAD(H), an essential cofactor in multiple reactions crucial for respiration, redox balance, and biosynthesis of major building blocks. NaMN adenylyltransferase (NadD) and NAD synthetase (NadE), the key enzymes of NAD biosynthesis, were selected as promising candidate drug targets for M. tuberculosis. Here we report for the first time kinetic characterization of the recombinant purified NadD enzyme, setting the stage for its structural analysis and inhibitor development. A protein knockdown approach was applied to validate bothNadD and NadE as target enzymes. Induced degradation of either target enzyme showed a strong bactericidal effect which coincided with anticipated changes in relative levels of NaMN and NaAD intermediates (substrates of NadD and NadE, respectively) and ultimate depletion of the NAD(H) pool. A metabolic catastrophe predicted as a likely result of NAD(H) deprivation of cellular metabolism was confirmed by 13C biosynthetic labeling followed by gas chromatography-mass spectrometry (GC-MS) analysis. A sharp suppression of metabolic flux was observed in multiple NAD(P)(H)-dependent pathways, including synthesis of many amino acids (serine, proline, aromatic amino acids) and fatty acids. Overall, these results provide strong validation of the essential NAD biosynthetic enzymes, NadD and NadE, as antimycobacterial drug targets.
IMPORTANCE
To address the problems of M. tuberculosis drug resistance and persistence of tuberculosis, new classes of drug targets need to be explored. The biogenesis of NAD cofactors was selected for target validation because of their indispensable role in driving hundreds of biochemical transformations. We hypothesized that the disruption of NAD production in the cell via genetic suppression of the essential enzymes (NadD and NadE) involved in the last two steps of NAD biogenesis would lead to cell death, even under dormancy conditions. In this study, we confirmed the hypothesis using a protein knockdown approach in the model system of Mycobacterium smegmatis. We showed that induced proteolytic degradation of either target enzyme leads to depletion of the NAD cofactor pool, which suppresses metabolic flux through numerous NAD(P)-dependent pathways of central metabolism of carbon and energy production. Remarkably, bactericidal effect was observed even for nondividing bacteria cultivated under carbon starvation conditions.
doi:10.1128/mBio.00747-13
PMCID: PMC3944813  PMID: 24549842
4.  Genetic determinants of in vivo fitness and diet responsiveness in multiple human gut Bacteroides 
Science (New York, N.Y.)  2015;350(6256):aac5992.
Libraries of tens of thousands of transposon mutants generated from each of four human gut Bacteroides strains, two representing the same species, were introduced simultaneously into gnotobiotic mice together with 11 other wild-type strains to generate a 15-member artificial human gut microbiota. Mice received one of two distinct diets monotonously, or both in ordered sequence. Quantifying the abundance of mutants in different diet contexts allowed gene-level characterization of fitness determinants, niche, stability and resilience, and yielded a prebiotic (arabinoxylan) that allowed targeted manipulation of the community. The approach described is generalizable and should be useful for defining mechanisms critical for sustaining and/or approaches for deliberately reconfiguring the highly adaptive and durable relationship between the human gut microbiota and host in ways that promote wellness.
doi:10.1126/science.aac5992
PMCID: PMC4608238  PMID: 26430127
5.  Quinolinate Salvage and Insights for Targeting NAD Biosynthesis in Group A Streptococci 
Journal of Bacteriology  2013;195(4):726-732.
The essential coenzyme NAD plays important roles in metabolic reactions and cell regulation in all organisms. As such, NAD synthesis has been investigated as a source for novel antibacterial targets. Cross-species genomics-based reconstructions of NAD metabolism in group A streptococci (GAS), combined with focused experimental testing in Streptococcus pyogenes, led to a better understanding of NAD metabolism in the pathogen. The predicted niacin auxotrophy was experimentally verified, as well as the essential role of the nicotinamidase PncA in the utilization of nicotinamide (Nm). PncA is dispensable in the presence of nicotinate (Na), ruling it out as a viable antibacterial target. The function of the “orphan” NadC enzyme, which is uniquely present in all GAS species despite the absence of other genes of NAD de novo synthesis, was elucidated. Indeed, the quinolinate (Qa) phosphoribosyltransferase activity of NadC from S. pyogenes allows the organism to sustain growth when Qa is present as a sole pyridine precursor. Finally, the redundancy of functional upstream salvage pathways in GAS species narrows the choice of potential drug targets to the two indispensable downstream enzymes of NAD synthesis, nicotinate adenylyltransferase (NadD family) and NAD synthetase (NadE family). Biochemical characterization of NadD confirmed its functional role in S. pyogenes, and its potential as an antibacterial target was supported by inhibition studies with previously identified class I inhibitors of the NadD enzyme family. One of these inhibitors efficiently inhibited S. pyogenes NadD (sp.NadD) in vitro (50% inhibitory concentration [IC50], 15 μM), exhibiting a noncompetitive mechanism with a Ki of 8 μM.
doi:10.1128/JB.02002-12
PMCID: PMC3562111  PMID: 23204464
6.  Regulation of Glutamine Carrier Proteins by RNF5 Determines Breast Cancer Response to ER Stress-inducing Chemotherapies 
Cancer cell  2015;27(3):354-369.
Summary
Many tumor cells are fueled by altered metabolism and increased glutamine (Gln) dependence. We identify regulation of the L-glutamine carrier proteins SLC1A5 and SLC38A2 (SLC1A5/38A2) by the ubiquitin ligase RNF5. Paclitaxel-induced ER stress to breast cancer (BCa) cells promotes RNF5 association, ubiquitination and degradation of SLC1A5/38A2. This decreases Gln uptake, levels of TCA cycle components, mTOR signaling and proliferation while increasing autophagy and cell death. Rnf5-deficient MMTV-PyMT mammary tumors were less differentiated and showed elevated SLC1A5 expression. Whereas RNF5 depletion in MDA-MB-231 cells promoted tumorigenesis and abolished paclitaxel responsiveness, SLC1A5/38A2 knockdown elicited opposing effects. Inverse RNF5HI/SLC1A5/38A2LO expression was associated with positive prognosis in BCa. Thus, RNF5 control of Gln uptake underlies BCa response to chemotherapies.
doi:10.1016/j.ccell.2015.02.006
PMCID: PMC4356903  PMID: 25759021
7.  Diversity and Versatility of the Thermotoga maritima Sugar Kinome 
Journal of Bacteriology  2012;194(20):5552-5563.
Sugar phosphorylation is an indispensable committed step in a large variety of sugar catabolic pathways, which are major suppliers of carbon and energy in heterotrophic species. Specialized sugar kinases that are indispensable for most of these pathways can be utilized as signature enzymes for the reconstruction of carbohydrate utilization machinery from microbial genomic and metagenomic data. Sugar kinases occur in several structurally distinct families with various partially overlapping as well as yet unknown substrate specificities that often cannot be accurately assigned by homology-based techniques. A subsystems-based metabolic reconstruction combined with the analysis of genome context and followed by experimental testing of predicted gene functions is a powerful approach of functional gene annotation. Here we applied this integrated approach for functional mapping of all sugar kinases constituting an extensive and diverse sugar kinome in the thermophilic bacterium Thermotoga maritima. Substrate preferences of 14 kinases mainly from the FGGY and PfkB families were inferred by bioinformatics analysis and biochemically characterized by screening with a panel of 45 different carbohydrates. Most of the analyzed enzymes displayed narrow substrate preferences corresponding to their predicted physiological roles in their respective catabolic pathways. The observed consistency supports the choice of kinases as signature enzymes for genomics-based identification and reconstruction of sugar utilization pathways. Use of the integrated genomic and experimental approach greatly speeds up the identification of the biochemical function of unknown proteins and improves the quality of reconstructed pathways.
doi:10.1128/JB.01136-12
PMCID: PMC3458674  PMID: 22885293
8.  Structural determinants of limited proteolysis 
Journal of proteome research  2011;10(8):3642-3651.
Limited or regulatory proteolysis plays a critical role in many important biological pathways like blood coagulation, cell proliferation, and apoptosis. A better understanding of mechanisms that control this process is required for discovering new proteolytic events and for developing inhibitors with potential therapeutic value. Two features that determine the susceptibility of peptide bonds to proteolysis are the sequence in the vicinity of the scissile bond and the structural context in which the bond is displayed. In this study we assessed statistical significance and predictive power of individual structural descriptors and combination thereof for the identification of cleavage sites. The analysis was performed on a dataset of >200 proteolytic events documented in CutDB for a variety of mammalian regulatory proteases and their physiological substrates with known 3D structures. The results confirmed the significance and provided a ranking within three main categories of structural features: exposure > flexibility > local interactions. Among secondary structure elements, the largest frequency of proteolytic cleavage was confirmed for loops and lower but significant frequency for helices. Limited proteolysis has lower albeit appreciable frequency of occurrence in certain types of β-strands, which is in contrast with some previous reports. Descriptors deduced directly from the amino acid sequence displayed only marginal predictive capabilities. Homology-based structural models showed a predictive performance comparable to protein substrates with experimentally established structures. Overall, this study provided a foundation for accurate automated prediction of segments of protein structure susceptible to proteolytic processing and, potentially, other post-translational modifications.
doi:10.1021/pr200271w
PMCID: PMC3164237  PMID: 21682278
proteolysis; proteolytic processing; limited proteolysis; regulatory proteolysis; protease; cleavage site; cleavage site prediction
9.  Experimental Strategies for Functional Annotation and Metabolism Discovery: Targeted Screening of Solute Binding Proteins and Unbiased Panning of Metabolomes 
Biochemistry  2014;54(3):909-931.
The rate at which genome sequencing data is accruing demands enhanced methods for functional annotation and metabolism discovery. Solute binding proteins (SBPs) facilitate the transport of the first reactant in a metabolic pathway, thereby constraining the regions of chemical space and the chemistries that must be considered for pathway reconstruction. We describe high-throughput protein production and differential scanning fluorimetry platforms, which enabled the screening of 158 SBPs against a 189 component library specifically tailored for this class of proteins. Like all screening efforts, this approach is limited by the practical constraints imposed by construction of the library, i.e., we can study only those metabolites that are known to exist and which can be made in sufficient quantities for experimentation. To move beyond these inherent limitations, we illustrate the promise of crystallographic- and mass spectrometric-based approaches for the unbiased use of entire metabolomes as screening libraries. Together, our approaches identified 40 new SBP ligands, generated experiment-based annotations for 2084 SBPs in 71 isofunctional clusters, and defined numerous metabolic pathways, including novel catabolic pathways for the utilization of ethanolamine as sole nitrogen source and the use of d-Ala-d-Ala as sole carbon source. These efforts begin to define an integrated strategy for realizing the full value of amassing genome sequence data.
doi:10.1021/bi501388y
PMCID: PMC4310620  PMID: 25540822
10.  Targeting NAD biosynthesis in bacterial pathogens. Structure-based development of inhibitors of nicotinate mononucleotide adenylyltransferase NadD 
Chemistry & biology  2009;16(8):849-861.
SUMMARY
The emergence of multidrug-resistant pathogens necessitates the search for new antibiotics acting on previously unexplored targets. Nicotinate mononucleotide adenylyltransferase of the NadD family, an essential enzyme of NAD biosynthesis in most bacteria, was selected as a target for structure-based inhibitor development. Using iterative in silico and in vitro screens we identified small molecule compounds that efficiently inhibited target enzymes from Escherichia coli (ecNadD) and Bacillus anthracis (baNadD) but had no effect on functionally equivalent human enzymes. On-target antibacterial activity was demonstrated for some of the selected inhibitors. A 3D structure of baNadD was solved in complex with one of these inhibitors (3_02) providing mechanistic insights and guidelines for further improvement. Most importantly, the results of this study help validate NadD as a target for the development of antibacterial agents with potential broad-spectrum activity.
doi:10.1016/j.chembiol.2009.07.006
PMCID: PMC2770502  PMID: 19716475
11.  Tagaturonate–fructuronate epimerase UxaE, a novel enzyme in the hexuronate catabolic network in Thermotoga maritima 
Environmental microbiology  2012;14(11):2920-2934.
Summary
Thermotoga maritima is a marine hyperthermophilic microorganism that degrades a wide range of simple and complex carbohydrates including pectin and produces fermentative hydrogen at high yield. Galacturonate and glucuronate, two abundant hexuronic acids in pectin and xylan, respectively, are catabolized via committed metabolic pathways to supply carbon and energy for a variety of microorganisms. By a combination of bioinformatics and experimental techniques we identified a novel enzyme family (named UxaE) catalysing a previously unknown reaction in the hexuronic acid catabolic pathway, epimerization of tagaturonate to fructuronate. The enzymatic activity of the purified recombinant tagaturonate epimerase from T. maritima was directly confirmed and kinetically characterized. Its function was also confirmed by genetic complementation of the growth of the Escherichia coli uxaB knockout mutant strain on galacturonate. An inferred novel galacturonate to mannonate catabolic pathway in T. maritima was reconstituted in vitro using a mixture of recombinant purified enzymes UxaE, UxaC and UxuB. Members of the newly identified UxaE family were identified in ~ 50 phylogenetically diverse heterotrophic bacteria from aquatic and soil environments. The genomic context of respective genes and reconstruction of associated pathways suggest that UxaE enzymatic and biological function remains conserved in all of these species.
doi:10.1111/j.1462-2920.2012.02856.x
PMCID: PMC4505799  PMID: 22925190
12.  An Agonist of Toll-Like Receptor 5 Has Radioprotective Activity in Mouse and Primate Models 
Science (New York, N.Y.)  2008;320(5873):226-230.
The toxicity of ionizing radiation is associated with massive apoptosis in radiosensitive organs. Here, we investigate whether a drug that activates a signaling mechanism used by tumor cells to suppress apoptosis can protect healthy cells from the harmful effects of radiation. We studied CBLB502, a polypeptide drug derived from Salmonella flagellin that binds to Toll-like receptor 5 (TLR5) and activates nuclear factor–κB signaling. A single injection of CBLB502 before lethal total-body irradiation protected mice from both gastrointestinal and hematopoietic acute radiation syndromes and resulted in improved survival. CBLB502 injected after irradiation also enhanced survival, but at lower radiation doses. It is noteworthy that the drug did not decrease tumor radiosensitivity in mouse models. CBLB502 also showed radioprotective activity in lethally irradiated rhesus monkeys. Thus, TLR5 agonists could potentially improve the therapeutic index of cancer radiotherapy and serve as biological protectants in radiation emergencies.
doi:10.1126/science.1154986
PMCID: PMC4322935  PMID: 18403709
13.  SEQUENCE-DERIVED STRUCTURAL FEATURES DRIVING PROTEOLYTIC PROCESSING 
Proteomics  2013;14(1):42-50.
Proteolytic signaling, or regulated proteolysis, is an essential part of many important pathways such as Notch, Wnt, and Hedgehog. How the structure of the cleaved substrate regions influences the efficacy of proteolytic processing remains underexplored. Here, we analyzed the relative importance in proteolysis of various structural features derived from substrate sequences using a dataset of more than five thousand experimentally verified proteolytic events captured in CutDB. Accessibility to the solvent was recognized as an essential property of a proteolytically processed polypeptide chain. Proteolytic events were found nearly uniformly distributed among three types of secondary structure, although with some enrichment in loops. Cleavages in α-helices were found to be relatively abundant in regions apparently prone to unfolding, while cleavages in β-structures tended to be located at the periphery of β-sheets. Application of the same statistical procedures to proteolytic events divided into separate sets according to the catalytic classes of proteases proved consistency of the results and confirmed that the structural mechanisms of proteolysis are universal. The estimated prediction power of sequence-derived structural features, which turned out to be sufficiently high, presents a rationale for their use in bioinformatic prediction of proteolytic events.
doi:10.1002/pmic.201300416
PMCID: PMC3926948  PMID: 24227478
14.  Comparative genomics and functional analysis of rhamnose catabolic pathways and regulons in bacteria 
L-rhamnose (L-Rha) is a deoxy-hexose sugar commonly found in nature. L-Rha catabolic pathways were previously characterized in various bacteria including Escherichia coli. Nevertheless, homology searches failed to recognize all the genes for the complete L-Rha utilization pathways in diverse microbial species involved in biomass decomposition. Moreover, the regulatory mechanisms of L-Rha catabolism have remained unclear in most species. A comparative genomics approach was used to reconstruct the L-Rha catabolic pathways and transcriptional regulons in the phyla Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Proteobacteria, and Thermotogae. The reconstructed pathways include multiple novel enzymes and transporters involved in the utilization of L-Rha and L-Rha-containing polymers. Large-scale regulon inference using bioinformatics revealed remarkable variations in transcriptional regulators for L-Rha utilization genes among bacteria. A novel bifunctional enzyme, L-rhamnulose-phosphate aldolase (RhaE) fused to L-lactaldehyde dehydrogenase (RhaW), which is not homologous to previously characterized L-Rha catabolic enzymes, was identified in diverse bacteria including Chloroflexi, Bacilli, and Alphaproteobacteria. By using in vitro biochemical assays we validated both enzymatic activities of the purified recombinant RhaEW proteins from Chloroflexus aurantiacus and Bacillus subtilis. Another novel enzyme of the L-Rha catabolism, L-lactaldehyde reductase (RhaZ), was identified in Gammaproteobacteria and experimentally validated by in vitro enzymatic assays using the recombinant protein from Salmonella typhimurium. C. aurantiacus induced transcription of the predicted L-Rha utilization genes when L-Rha was present in the growth medium and consumed L-Rha from the medium. This study provided comprehensive insights to L-Rha catabolism and its regulation in diverse Bacteria.
doi:10.3389/fmicb.2013.00407
PMCID: PMC3870299  PMID: 24391637
L-rhamnose catabolism; metabolic reconstruction; regulon; comparative genomics; Chloroflexus
15.  Polysaccharides utilization in human gut bacterium Bacteroides thetaiotaomicron: comparative genomics reconstruction of metabolic and regulatory networks 
BMC Genomics  2013;14:873.
Background
Bacteroides thetaiotaomicron, a predominant member of the human gut microbiota, is characterized by its ability to utilize a wide variety of polysaccharides using the extensive saccharolytic machinery that is controlled by an expanded repertoire of transcription factors (TFs). The availability of genomic sequences for multiple Bacteroides species opens an opportunity for their comparative analysis to enable characterization of their metabolic and regulatory networks.
Results
A comparative genomics approach was applied for the reconstruction and functional annotation of the carbohydrate utilization regulatory networks in 11 Bacteroides genomes. Bioinformatics analysis of promoter regions revealed putative DNA-binding motifs and regulons for 31 orthologous TFs in the Bacteroides. Among the analyzed TFs there are 4 SusR-like regulators, 16 AraC-like hybrid two-component systems (HTCSs), and 11 regulators from other families. Novel DNA motifs of HTCSs and SusR-like regulators in the Bacteroides have the common structure of direct repeats with a long spacer between two conserved sites.
Conclusions
The inferred regulatory network in B. thetaiotaomicron contains 308 genes encoding polysaccharide and sugar catabolic enzymes, carbohydrate-binding and transport systems, and TFs. The analyzed TFs control pathways for utilization of host and dietary glycans to monosaccharides and their further interconversions to intermediates of the central metabolism. The reconstructed regulatory network allowed us to suggest and refine specific functional assignments for sugar catabolic enzymes and transporters, providing a substantial improvement to the existing metabolic models for B. thetaiotaomicron. The obtained collection of reconstructed TF regulons is available in the RegPrecise database (http://regprecise.lbl.gov).
doi:10.1186/1471-2164-14-873
PMCID: PMC3878776  PMID: 24330590
Regulatory network; Regulon; Transcription factor; BACTEROIDES; Carbohydrate utilization
16.  Glutamine-fueled mitochondrial metabolism is decoupled from glycolysis in melanoma 
Pigment cell & melanoma research  2012;25(6):732-739.
Summary
In this perspective, we revise the historic notion that cancer is a disease of mitochondria. We summarize recent findings on the function and rewiring of central carbon metabolism in melanoma. Metabolic profiling studies using stable isotope tracers show that glycolysis is decoupled from the tricarboxylic acid (TCA) cycle. This decoupling is not ‘dysfunction’ but rather an alternate wiring required by tumor cells to remain metabolically versatile. In large part, this requirement is met by glutamine feeding the TCA cycle as an alternative source of carbon. Glutamine is also used in non-conventional ways, like traveling in reverse through the TCA flux to feed fatty acid biosynthesis. The biosynthetic networks linked with non-essential amino acids alanine, serine, arginine, and proline are also significantly impacted by the use of glutamine as an alternate carbon source.
doi:10.1111/pcmr.12000
PMCID: PMC3639292  PMID: 22846158
metabolism; mitochondria; glutamine; systems biology; NMR
17.  The COMBREX Project: Design, Methodology, and Initial Results 
Anton, Brian P. | Chang, Yi-Chien | Brown, Peter | Choi, Han-Pil | Faller, Lina L. | Guleria, Jyotsna | Hu, Zhenjun | Klitgord, Niels | Levy-Moonshine, Ami | Maksad, Almaz | Mazumdar, Varun | McGettrick, Mark | Osmani, Lais | Pokrzywa, Revonda | Rachlin, John | Swaminathan, Rajeswari | Allen, Benjamin | Housman, Genevieve | Monahan, Caitlin | Rochussen, Krista | Tao, Kevin | Bhagwat, Ashok S. | Brenner, Steven E. | Columbus, Linda | de Crécy-Lagard, Valérie | Ferguson, Donald | Fomenkov, Alexey | Gadda, Giovanni | Morgan, Richard D. | Osterman, Andrei L. | Rodionov, Dmitry A. | Rodionova, Irina A. | Rudd, Kenneth E. | Söll, Dieter | Spain, James | Xu, Shuang-yong | Bateman, Alex | Blumenthal, Robert M. | Bollinger, J. Martin | Chang, Woo-Suk | Ferrer, Manuel | Friedberg, Iddo | Galperin, Michael Y. | Gobeill, Julien | Haft, Daniel | Hunt, John | Karp, Peter | Klimke, William | Krebs, Carsten | Macelis, Dana | Madupu, Ramana | Martin, Maria J. | Miller, Jeffrey H. | O'Donovan, Claire | Palsson, Bernhard | Ruch, Patrick | Setterdahl, Aaron | Sutton, Granger | Tate, John | Yakunin, Alexander | Tchigvintsev, Dmitri | Plata, Germán | Hu, Jie | Greiner, Russell | Horn, David | Sjölander, Kimmen | Salzberg, Steven L. | Vitkup, Dennis | Letovsky, Stanley | Segrè, Daniel | DeLisi, Charles | Roberts, Richard J. | Steffen, Martin | Kasif, Simon
PLoS Biology  2013;11(8):e1001638.
Experimental data exists for only a vanishingly small fraction of sequenced microbial genes. This community page discusses the progress made by the COMBREX project to address this important issue using both computational and experimental resources.
doi:10.1371/journal.pbio.1001638
PMCID: PMC3754883  PMID: 24013487
18.  Transcriptional regulation of the carbohydrate utilization network in Thermotoga maritima 
Hyperthermophilic bacteria from the Thermotogales lineage can produce hydrogen by fermenting a wide range of carbohydrates. Previous experimental studies identified a large fraction of genes committed to carbohydrate degradation and utilization in the model bacterium Thermotoga maritima. Knowledge of these genes enabled comprehensive reconstruction of biochemical pathways comprising the carbohydrate utilization network. However, transcriptional factors (TFs) and regulatory mechanisms driving this network remained largely unknown. Here, we used an integrated approach based on comparative analysis of genomic and transcriptomic data for the reconstruction of the carbohydrate utilization regulatory networks in 11 Thermotogales genomes. We identified DNA-binding motifs and regulons for 19 orthologous TFs in the Thermotogales. The inferred regulatory network in T. maritima contains 181 genes encoding TFs, sugar catabolic enzymes and ABC-family transporters. In contrast to many previously described bacteria, a transcriptional regulation strategy of Thermotoga does not employ global regulatory factors. The reconstructed regulatory network in T. maritima was validated by gene expression profiling on a panel of mono- and disaccharides and by in vitro DNA-binding assays. The observed upregulation of genes involved in catabolism of pectin, trehalose, cellobiose, arabinose, rhamnose, xylose, glucose, galactose, and ribose showed a strong correlation with the UxaR, TreR, BglR, CelR, AraR, RhaR, XylR, GluR, GalR, and RbsR regulons. Ultimately, this study elucidated the transcriptional regulatory network and mechanisms controlling expression of carbohydrate utilization genes in T. maritima. In addition to improving the functional annotations of associated transporters and catabolic enzymes, this research provides novel insights into the evolution of regulatory networks in Thermotogales.
doi:10.3389/fmicb.2013.00244
PMCID: PMC3750489  PMID: 23986752
carbohydrate metabolism; transcriptional regulation; regulon; comparative genomics; Thermotoga
19.  Genomics-Guided Analysis of NAD Recycling Yields Functional Elucidation of COG1058 as a New Family of Pyrophosphatases 
PLoS ONE  2013;8(6):e65595.
We have recently identified the enzyme NMN deamidase (PncC), which plays a key role in the regeneration of NAD in bacteria by recycling back to the coenzyme the pyridine by-products of its non redox consumption. In several bacterial species, PncC is fused to a COG1058 domain of unknown function, highly conserved and widely distributed in all living organisms. Here, we demonstrate that the PncC-fused domain is endowed with a novel Co+2- and K+-dependent ADP-ribose pyrophosphatase activity, and discuss the functional connection of such an activity with NAD recycling. An in-depth phylogenetic analysis of the COG1058 domain evidenced that in most bacterial species it is fused to PncC, while in α- and some δ-proteobacteria, as well as in archaea and fungi, it occurs as a stand-alone protein. Notably, in mammals and plants it is fused to FAD synthase. We extended the enzymatic characterization to a representative bacterial single-domain protein, which resulted to be a more versatile ADP-ribose pyrophosphatase, active also towards diadenosine 5′-diphosphate and FAD. Multiple sequence alignment analysis, and superposition of the available three-dimensional structure of an archaeal COG1058 member with the structure of the enzyme MoeA of the molybdenum cofactor biosynthesis, allowed identification of residues likely involved in catalysis. Their role has been confirmed by site-directed mutagenesis.
doi:10.1371/journal.pone.0065595
PMCID: PMC3680494  PMID: 23776507
20.  Reverse TCA cycle flux through isocitrate dehydrogenases 1 and 2 is required for lipogenesis in hypoxic melanoma cells 
Pigment cell & melanoma research  2012;25(3):375-383.
Summary
The TCA cycle is the central hub of oxidative metabolism, running in the classic forward direction to provide carbon for biosynthesis and reducing agents for generation of ATP. Our metabolic tracer studies in melanoma cells showed that in hypoxic conditions the TCA cycle is largely disconnected from glycolysis. By studying the TCA branch point metabolites, acetyl CoA and citrate, as well as the metabolic endpoints glutamine and fatty acids, we developed a comprehensive picture of the rewiring of the TCA cycle that occurs in hypoxia. Hypoxic tumor cells maintain proliferation by running the TCA cycle in reverse. The source of carbon for acetyl CoA, citrate, and fatty acids switches from glucose in normoxia to glutamine in hypoxia. This hypoxic flux from glutamine into fatty acids is mediated by reductive carboxylation. This reductive carboxylation is catalyzed by two isocitrate dehydrogenases, IDH1 and IDH2. Their combined action is necessary and sufficient to effect the reverse TCA flux and maintain cellular viability.
doi:10.1111/j.1755-148X.2012.00989.x
PMCID: PMC3329592  PMID: 22360810
21.  Structural basis of TLR5-flagellin recognition and signaling 
Science (New York, N.Y.)  2012;335(6070):859-864.
Toll-like receptor 5 (TLR5) binding to bacterial flagellin activates NF-κB signaling and triggers an innate immune response to the invading pathogen. To elucidate the structural basis and mechanistic implications of TLR5-flagellin recognition, we determined the crystal structure of zebrafish TLR5, as a VLR-hybrid protein, in complex with the D1/D2 fragment of Salmonella flagellin, FliC, at 2.47 Å resolution. TLR5 interacts primarily with the three helices of the FliC D1 domain using its lateral side. Two TLR5-FliC 1:1 heterodimers assemble into a 2:2 tail-to-tail signaling complex that is stabilized by quaternary contacts of the FliC D1 domain with the convex surface of the opposing TLR5. The proposed signaling mechanism is supported by structure-guided mutagenesis and deletion analysis on CBLB502, a therapeutic protein derived from FliC.
doi:10.1126/science.1215584
PMCID: PMC3406927  PMID: 22344444
22.  Functional diversification of ROK-family transcriptional regulators of sugar catabolism in the Thermotogae phylum 
Nucleic Acids Research  2012;41(2):790-803.
Large and functionally heterogeneous families of transcription factors have complex evolutionary histories. What shapes specificities toward effectors and DNA sites in paralogous regulators is a fundamental question in biology. Bacteria from the deep-branching lineage Thermotogae possess multiple paralogs of the repressor, open reading frame, kinase (ROK) family regulators that are characterized by carbohydrate-sensing domains shared with sugar kinases. We applied an integrated genomic approach to study functions and specificities of regulators from this family. A comparative analysis of 11 Thermotogae genomes revealed novel mechanisms of transcriptional regulation of the sugar utilization networks, DNA-binding motifs and specific functions. Reconstructed regulons for seven groups of ROK regulators were validated by DNA-binding assays using purified recombinant proteins from the model bacterium Thermotoga maritima. All tested regulators demonstrated specific binding to their predicted cognate DNA sites, and this binding was inhibited by specific effectors, mono- or disaccharides from their respective sugar catabolic pathways. By comparing ligand-binding domains of regulators with structurally characterized kinases from the ROK family, we elucidated signature amino acid residues determining sugar-ligand regulator specificity. Observed correlations between signature residues and the sugar-ligand specificities provide the framework for structure functional classification of the entire ROK family.
doi:10.1093/nar/gks1184
PMCID: PMC3553997  PMID: 23209028
23.  Functional Specialization in Proline Biosynthesis of Melanoma 
PLoS ONE  2012;7(9):e45190.
Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C), and then to proline via pyrroline-5-carboxylate reductases (PYCRs). Here, the role of three isozymic versions of PYCR was addressed in human melanoma cells by tracking the fate of 13C-labeled precursors. Based on these studies we conclude that PYCR1 and PYCR2, which are localized in the mitochondria, are primarily involved in conversion of glutamate to proline. PYCRL, localized in the cytosol, is exclusively linked to the conversion of ornithine to proline. This analysis provides the first clarification of the role of PYCRs to proline biosynthesis.
doi:10.1371/journal.pone.0045190
PMCID: PMC3443215  PMID: 23024808
24.  Transcriptional Regulation of Central Carbon and Energy Metabolism in Bacteria by Redox-Responsive Repressor Rex 
Journal of Bacteriology  2012;194(5):1145-1157.
Redox-sensing repressor Rex was previously implicated in the control of anaerobic respiration in response to the cellular NADH/NAD+ levels in Gram-positive bacteria. We utilized the comparative genomics approach to infer candidate Rex-binding DNA motifs and assess the Rex regulon content in 119 genomes from 11 taxonomic groups. Both DNA-binding and NAD-sensing domains are broadly conserved in Rex orthologs identified in the phyla Firmicutes, Thermotogales, Actinobacteria, Chloroflexi, Deinococcus-Thermus, and Proteobacteria. The identified DNA-binding motifs showed significant conservation in these species, with the only exception detected in Clostridia, where the Rex motif deviates in two positions from the generalized consensus, TTGTGAANNNNTTCACAA. Comparative analysis of candidate Rex sites revealed remarkable variations in functional repertoires of candidate Rex-regulated genes in various microorganisms. Most of the reconstructed regulatory interactions are lineage specific, suggesting frequent events of gain and loss of regulator binding sites in the evolution of Rex regulons. We identified more than 50 novel Rex-regulated operons encoding functions that are essential for resumption of the NADH:NAD+ balance. The novel functional role of Rex in the control of the central carbon metabolism and hydrogen production genes was validated by in vitro DNA binding assays using the TM0169 protein in the hydrogen-producing bacterium Thermotoga maritima.
doi:10.1128/JB.06412-11
PMCID: PMC3294762  PMID: 22210771
25.  Glutamine versus Ammonia Utilization in the NAD Synthetase Family 
PLoS ONE  2012;7(6):e39115.
NAD is a ubiquitous and essential metabolic redox cofactor which also functions as a substrate in certain regulatory pathways. The last step of NAD synthesis is the ATP-dependent amidation of deamido-NAD by NAD synthetase (NADS). Members of the NADS family are present in nearly all species across the three kingdoms of Life. In eukaryotic NADS, the core synthetase domain is fused with a nitrilase-like glutaminase domain supplying ammonia for the reaction. This two-domain NADS arrangement enabling the utilization of glutamine as nitrogen donor is also present in various bacterial lineages. However, many other bacterial members of NADS family do not contain a glutaminase domain, and they can utilize only ammonia (but not glutamine) in vitro. A single-domain NADS is also characteristic for nearly all Archaea, and its dependence on ammonia was demonstrated here for the representative enzyme from Methanocaldococcus jannaschi. However, a question about the actual in vivo nitrogen donor for single-domain members of the NADS family remained open: Is it glutamine hydrolyzed by a committed (but yet unknown) glutaminase subunit, as in most ATP-dependent amidotransferases, or free ammonia as in glutamine synthetase? Here we addressed this dilemma by combining evolutionary analysis of the NADS family with experimental characterization of two representative bacterial systems: a two-subunit NADS from Thermus thermophilus and a single-domain NADS from Salmonella typhimurium providing evidence that ammonia (and not glutamine) is the physiological substrate of a typical single-domain NADS. The latter represents the most likely ancestral form of NADS. The ability to utilize glutamine appears to have evolved via recruitment of a glutaminase subunit followed by domain fusion in an early branch of Bacteria. Further evolution of the NADS family included lineage-specific loss of one of the two alternative forms and horizontal gene transfer events. Lastly, we identified NADS structural elements associated with glutamine-utilizing capabilities.
doi:10.1371/journal.pone.0039115
PMCID: PMC3376133  PMID: 22720044

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