In contrast to 5-methylcytosine (5-mC), which has been studied extensively1–3, little is known about 5-hydroxymethylcytosine (5-hmC), a recently identified epigenetic modification present in substantial amounts in certain mammalian cell types4,5. Here we present a method for determining the genome-wide distribution of 5-hmC. We use the T4 bacteriophage β-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC. The azide group can be chemically modified with biotin for detection, affinity enrichment and sequencing of 5-hmC–containing DNA fragments in mammalian genomes. Using this method, we demonstrate that 5-hmC is present in human cell lines beyond those previously recognized4. We also find a gene expression level–dependent enrichment of intragenic 5-hmC in mouse cerebellum and an age-dependent acquisition of this modification in specific gene bodies linked to neurodegenerative disorders.

Antimicrobial drug resistance is an urgent problem in control and treatment of many of the world's most serious infections, including Plasmodium falciparum malaria, tuberculosis, and healthcare-associated infections with Gram-negative bacteria. Because the non-mevalonate pathway of isoprenoid biosynthesis is essential in eubacteria and P. falciparum, and this pathway is not present in humans, there is great interest in targeting the enzymes of non-mevalonate metabolism for antibacterial and antiparasitic drug development. Fosmidomycin is a broad-spectrum antimicrobial agent currently in clinical trials of combination therapies to treat malaria. In vitro, fosmidomycin is known to inhibit the deoxyxylulose phosphate reductoisomerase (DXR) enzyme of isoprenoid biosynthesis from multiple pathogenic organisms. To define the in vivo metabolic response to fosmidomycin, we developed a novel mass spectrometry method to quantitate six metabolites of non-mevalonate isoprenoid metabolism from complex biological samples. Using this technique, we validate that the biological effects of fosmidomycin are mediated through blockade of de novo isoprenoid biosynthesis in both P. falciparum malaria parasites and E. coli bacteria: in both organisms, metabolic profiling demonstrated a block in isoprenoid metabolism following fosmidomycin treatment, and growth inhibition due to fosmidomycin was rescued by media supplemented with isoprenoid metabolites. Isoprenoid metabolism proceeded through DXR even in the presence of fosmidomycin, but was inhibited at the level of the downstream enzyme, methylerythritol phosphate cytidyltransferase (IspD). Overexpression of IspD in E. coli conferred fosmidomycin resistance, and fosmidomycin was found to inhibit IspD in vitro. This work has validated fosmidomycin as a biological reagent to block non-mevalonate isoprenoid metabolism, and suggests a second in vivo target for fosmidomycin within isoprenoid biosynthesis, in two evolutionarily diverse pathogens.

Cucurbitaceous plants (cucurbits) have long been preferred models for studying phloem physiology. However, these species are unusual in that they possess two different phloem systems, one within the main vascular bundles [fascicular phloem (FP)] and another peripheral to the vascular bundles and scattered through stem and petiole cortex tissues [extrafascicular phloem (EFP)]. We have revisited the assumption that the sap released after shoot incision originates from the FP, and also investigated the long-standing question of why the sugar content of this sap is ∼30-fold less than predicted for requirements of photosynthate delivery. Video microscopy and phloem labeling experiments unexpectedly reveal that FP very quickly becomes blocked upon cutting, whereas the extrafascicular phloem bleeds for extended periods. Thus, all cucurbit phloem sap studies to date have reported metabolite, protein, and RNA composition and transport in the relatively minor extrafascicular sieve tubes. Using tissue dissection and direct sampling of sieve tube contents, we show that FP in fact does contain up to 1 M sugars, in contrast to low-millimolar levels in the EFP. Moreover, major phloem proteins in sieve tubes of FP differ from those that predominate in the extrafascicular sap, and include several previously uncharacterized proteins with little or no homology to databases. The overall compositional differences of the two phloem systems strongly indicate functional isolation. On this basis, we propose that the fascicular phloem is largely responsible for sugar transport, whereas the extrafascicular phloem may function in signaling, defense, and transport of other metabolites.cucurbitaceae | symplastic loading | extrafascicular | exudate | P-protein

SUMMARYThe rice gene ELONGATED UPPERMOST INTERNODE1 (EUI1) encodes a P450 monooxygenase that epoxidizes gibberellins (GAs) in a deactivation reaction. The Arabidopsis genome contains a tandemly duplicated gene pair ELA1 (CYP714A1) and ELA2 (CYP714A2) that encode EUI homologs. In this work, we dissected the functions of the two proteins. ELA1 and ELA2 exhibited overlapping yet distinct gene expression patterns. We showed that while single mutants of ELA1 or ELA2 exhibited no obvious morphological phenotype, simultaneous elimination of ELA1 and ELA2 expression in ELA1-RNAi/ela2 resulted in increased biomass and enlarged organs. By contrast, transgenic plants constitutively expressing either ELA1 or ELA2 were dwarfed, similar to those overexpressing the rice EUI gene. We also discovered that overexpression of ELA1 resulted in a severe dwarf phenotype, while overexpression of ELA2 gave rise to a breeding-favored semi-dwarf phenotype in rice. Consistent with the phenotypes, we found that the ELA1-RNAi/ela2 plants increased amounts of biologically active GAs that were decreased in the internodes of transgenic rice with ELA1 and ELA2 overexpression. In contrast, the precursor GA 12 slightly accumulated in the transgenic rice, and GA 19 highly accumulated in the ELA2 overexpression rice. Taken together, our study strongly suggests that the two Arabidopsis EUI homologs subtly regulate plant growth most likely through catalyzing deactivation of bioactive GAs similar to rice EUI. The two P450s may also function in early stages of the GA biosynthetic pathway. Our results also suggest that ELA2 could be an excellent tool for molecular breeding for high yield potential in cereal crops.

Thermoanaerobacter sp. strain X514 has great potential in biotechnology due to its capacity to ferment a range of C 5 and C 6 sugars to ethanol and other metabolites under thermophilic conditions. This study investigated the central metabolism of strain X514 via 13 C-labeled tracer experiments using either glucose or pyruvate as both carbon and energy sources. X514 grew on minimal medium and thus contains complete biosynthesis pathways for all macromolecule building blocks. Based on genome annotation and isotopic analysis of amino acids, three observations can be obtained about the central metabolic pathways in X514. First, the oxidative pentose phosphate pathway in X514 is not functional, and the tricarboxylic acid cycle is incomplete under fermentative growth conditions. Second, X514 contains (Re)-type citrate synthase activity, although no gene homologous to the recently characterized (Re)-type citrate synthase of Clostridium kluyveri was found. Third, the isoleucine in X514 is derived from acetyl coenzyme A and pyruvate via the citramalate pathway rather than being synthesized from threonine via threonine ammonia-lyase. The functionality of the citramalate synthase gene (cimA [Teth514_1204]) has been confirmed by enzymatic activity assays, while the presence of intracellular citramalate has been detected by mass spectrometry. This study demonstrates the merits of combining 13 C-assisted metabolite analysis, enzyme assays, and metabolite detection not only to examine genome sequence annotations but also to discover novel enzyme activities.

Cytochrome P450 (P450) 2 enzymes catalyze the most versatile chemical reactions in nature (1). There is, however, a discrepancy between the plant and the animal kingdoms with regard to the sheer number of these biocatalysts. Whereas in a single model plant, Arabidopsis thaliana, there are to date 273 P450 proteins, in the human genome, only 57 of these proteins are present. Whereas plants and animals share a multitude of highly regio-and stereospecific O-demethylation reactions, more complex reactions such as phenol coupling are much more abundant in plants than in animals, especially in the alkaloid field (2-11). The proposal of Barton and Cohen (12) correlated the structure of specific plant alkaloids in terms of this reaction mechanism and gave mechanistic proposals of how these phenol-coupled products may possibly be biosynthesized in nature. The oxidation of phenols by one-electron transfer affords radicals, which, by radical pairing, form new C-C or C-O bonds either by intra-or intermolecular coupling. The first two examples that unequivocally demonstrated the formation of C-C and C-O bonds in a stereo-and regioselective manner in plant metabolism are catalyzed by specific P450-linked microsomal-bound plant enzymes (13). One of these enzymes was salutaridine synthase from Papaver somniferum (opium poppy) (14). This synthase catalyzes the intramolecular formation of the critical C12-C13 carbon bridge and is a key enzyme in morphine biosynthesis.The groups of Goldstein (15) and Spector (16) have published a number of reports over the past 25 years claiming that mammals are capable of synthesizing de novo traces of endogenous morphine. However, no convincing experimental data have been presented regarding the enzymes. Phenol-coupling reactions in mammals are extremely rare, and the only example described thus far is the formation of thyroxine, by radical pairing, in humans. If the key step of morphine synthesis, the formation of phenol-coupled salutaridine from (R)-reticuline, occurs in mammals then in analogy to plants, a P450 enzyme must be present (in mammals) to catalyze this reaction. In 1987, the first experiments were conducted in an attempt to discover the reaction by supplying uniformly labeled racemic [ 3 H]reticuline in the presence of rat microsomes and NADPH to examine whether [ 3 H]salutaridine can be formed under these conditions (15). A radioactive compound was formed in 1% yield and assumed to be the phenol-coupled product salutaridine. We later repeated this experiment using (R)-[N-14 CH 3 ]reticuline and NADPH-fortified microsomes from pig, rat, cow, and sheep. We observed the formation of [N-14 CH 3 ]salutaridine with the correct stereochemistry at carbon 9 (17). The enzyme from porcine liver was subsequently purified to homogeneity and the reaction product was characterized by mass spectrometry and physical parameters to be a product of a P450 enzyme, which we provisionally named "salutaridine synthase" (18). The aim of this report is the identification of the homogenous porcine P450 e...

The applications of stable isotopes in metabolomics have facilitated the study of cell metabolisms. Stable isotope-assisted metabolomics requires: (1) properly designed tracer experiments; (2) stringent sampling and quenching protocols to minimize isotopic alternations; (3) efficient metabolite separations; (4) high resolution mass spectrometry to resolve overlapping peaks and background noises; and (5) data analysis methods and databases to decipher isotopic clusters over a broad m/z range (mass-to-charge ratio). This paper overviews mass spectrometry based techniques for precise determination of metabolites and their isotopologues. It also discusses applications of isotopic approaches to track substrate utilization, identify unknown metabolites and their chemical formulas, measure metabolite concentrations, determine putative metabolic pathways, and investigate microbial community populations and their carbon assimilation patterns. In addition, 13C-metabolite fingerprinting and metabolic models can be integrated to quantify carbon fluxes (enzyme reaction rates). The fluxome, in combination with other “omics” analyses, may give systems-level insights into regulatory mechanisms underlying gene functions. More importantly, 13C-tracer experiments significantly improve the potential of low-resolution gas chromatography-mass spectrometry (GC-MS) for broad-scope metabolism studies. We foresee the isotope-assisted metabolomics to be an indispensable tool in industrial biotechnology, environmental microbiology, and medical research.

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