Functionally redundant formate dehydrogenases enable formate-dependent growth in Methanococcus maripaludis.

Methanogens are essential for the complete remineralization of organic matter in anoxic environments. Most cultured methanogens are hydrogenotrophic, using H2 as an electron donor to reduce CO2 to CH4, but in the absence of H2 many can also use formate. Formate dehydrogenase (Fdh) is essential for formate oxidation, where it transfers electrons for the reduction of coenzyme F420 or to a flavin-based electron bifurcating reaction catalyzed by heterodisulfide reductase (Hdr), the terminal reaction of methanogenesis. Furthermore, methanogens that use formate encode at least two isoforms of Fdh in their genomes, but how these different isoforms participate in methanogenesis is unknown. Using Methanococcus maripaludis, we undertook a biochemical characterization of both Fdh isoforms involved in methanogenesis. Both Fdh1 and Fdh2 interacted with Hdr to catalyze the flavin-based electron bifurcating reaction, and both reduced F420 at similar rates. F420 reduction preceded flavin-based electron bifurcation activity for both enzymes. In a Δfdh1 mutant background, a suppressor mutation was required for Fdh2 activity. Genome sequencing revealed that this mutation resulted in the loss of a specific molybdopterin transferase (moeA), allowing for Fdh2-dependent growth, and the metal content of the proteins suggested that isoforms are dependent on either molybdenum or tungsten for activity. These data suggest that both isoforms of Fdh are functionally redundant, but their activities in vivo may be limited by gene regulation or metal availability under different growth conditions. Together these results expand our understanding of formate oxidation and the role of Fdh in methanogenesis.

Identification of a S-(2-succino)cysteine breakdown pathway that uses a novel S-(2-succino) lyase.

Succination is the spontaneous reaction between the respiratory intermediate fumarate and cellular thiols that forms stable S-(2-succino)-adducts such as S-(2-succino)cysteine (2SC). 2SC is a biomarker for conditions associated with elevated fumarate levels, including diabetes, obesity, and certain cancers, and succination likely contributes to disease progression. Bacillus subtilis has a yxe operon-encoded breakdown pathway for 2SC that involves three distinct enzymatic conversions. The first step is N-acetylation of 2SC by YxeL to form N-acetyl-2SC (2SNAC). YxeK catalyzes the oxygenation of 2SNAC, resulting in its breakdown to oxaloacetate and N-acetylcysteine, which is deacetylated by YxeP to give cysteine. The monooxygenase YxeK is key to the pathway but is rare, with close homologs occurring infrequently in prokaryote and fungal genomes. The existence of additional 2SC breakdown pathways was not known prior to this study. Here, we used comparative genomics to identify a S-(2-succino) lyase (2SL) that replaces yxeK in some yxe gene clusters. 2SL genes from Enterococcus italicus and Dickeya dadantii complement B. subtilis yxeK mutants. We also determined that recombinant 2SL enzymes efficiently break down 2SNAC into fumarate and N-acetylcysteine, can perform the reverse reaction, and have minor activity against 2SC and other small molecule thiols. The strong preferences both YxeK and 2SL enzymes have for 2SNAC indicate that 2SC acetylation is a conserved breakdown step. The identification of a second naturally occurring 2SC breakdown pathway underscores the importance of 2SC catabolism and defines a general strategy for 2SC breakdown involving acetylation, breakdown, and deacetylation.

Chemical-damage MINE: A database of curated and predicted spontaneous metabolic reactions.

Spontaneous reactions between metabolites are often neglected in favor of emphasizing enzyme-catalyzed chemistry because spontaneous reaction rates are assumed to be insignificant under physiological conditions. However, synthetic biology and engineering efforts can raise natural metabolites' levels or introduce unnatural ones, so that previously innocuous or nonexistent spontaneous reactions become an issue. Problems arise when spontaneous reaction rates exceed the capacity of a platform organism to dispose of toxic or chemically active reaction products. While various reliable sources list competing or toxic enzymatic pathways' side-reactions, no corresponding compilation of spontaneous side-reactions exists, nor is it possible to predict their occurrence. We addressed this deficiency by creating the Chemical Damage (CD)-MINE resource. First, we used literature data to construct a comprehensive database of metabolite reactions that occur spontaneously in physiological conditions. We then leveraged this data to construct 148 reaction rules describing the known spontaneous chemistry in a substrate-generic way. We applied these rules to all compounds in the ModelSEED database, predicting 180,891 spontaneous reactions. The resulting (CD)-MINE is available at https://minedatabase.mcs.anl.gov/cdmine/#/home and through developer tools. We also demonstrate how damage-prone intermediates and end products are widely distributed among metabolic pathways, and how predicting spontaneous chemical damage helps rationalize toxicity and carbon loss using examples from published pathways to commercial products. We explain how analyzing damage-prone areas in metabolism helps design effective engineering strategies. Finally, we use the CD-MINE toolset to predict the formation of the novel damage product N-carbamoyl proline, and present mass spectrometric evidence for its presence in Escherichia coli.

PioABC-Dependent Fe(II) Oxidation during Photoheterotrophic Growth on an Oxidized Carbon Substrate Increases Growth Yield.

Microorganisms that carry out Fe(II) oxidation play a major role in biogeochemical cycling of iron in environments with low oxygen. Fe(II) oxidation has been largely studied in the context of autotrophy. Here, we show that the anoxygenic phototroph, Rhodopseudomonas palustris CGA010, carries out Fe(II) oxidation during photoheterotrophic growth with an oxidized carbon source, malate, leading to an increase in cell yield and allowing more carbon to be directed to cell biomass. We probed the regulatory basis for this by transcriptome sequencing (RNA-seq) and found that the expression levels of the known pioABC Fe(II) oxidation genes in R. palustris depended on the redox-sensing two-component system, RegSR, and the oxidation state of the carbon source provided to cells. This provides the first mechanistic demonstration of mixotrophic growth involving reducing power generated from both Fe(II) oxidation and carbon assimilation. IMPORTANCE The simultaneous use of carbon and reduced metals such as Fe(II) by bacteria is thought to be widespread in aquatic environments, and a mechanistic description of this process could improve our understanding of biogeochemical cycles. Anoxygenic phototrophic bacteria like Rhodopseudomonas palustris typically use light for energy and organic compounds as both a carbon and an electron source. They can also use CO2 for carbon by carbon dioxide fixation when electron-rich compounds like H2, thiosulfate, and Fe(II) are provided as electron donors. Here, we show that Fe(II) oxidation can be used in another context to promote higher growth yields of R. palustris when the oxidized carbon compound malate is provided. We further established the regulatory mechanism underpinning this observation.

The metabolite repair enzyme Nit1 is a dual-targeted amidase that disposes of damaged glutathione in Arabidopsis.

The tripeptide glutathione (GSH) is implicated in various crucial physiological processes including redox buffering and protection against heavy metal toxicity. GSH is abundant in plants, with reported intracellular concentrations typically in the 1-10 mM range. Various aminotransferases can inadvertently transaminate the amino group of the γ-glutamyl moiety of GSH to produce deaminated glutathione (dGSH), a metabolite damage product. It was recently reported that an amidase known as Nit1 participates in dGSH breakdown in mammals and yeast. Plants have a hitherto uncharacterized homolog of the Nit1 amidase. We show that recombinant Arabidopsis Nit1 (At4g08790) has high and specific amidase activity towards dGSH. Ablating the Arabidopsis Nit1 gene causes a massive accumulation of dGSH and other marked changes to the metabolome. All plant Nit1 sequences examined had predicted plastidial targeting peptides with a potential second start codon whose use would eliminate the targeting peptide. In vitro transcription/translation assays show that both potential translation start codons in Arabidopsis Nit1 were used and confocal microscopy of Nit1-GFP fusions in plant cells confirmed both cytoplasmic and plastidial localization. Furthermore, we show that Arabidopsis enzymes present in leaf extracts convert GSH to dGSH at a rate of 2.8 pmol min-1 mg-1 in the presence of glyoxalate as an amino acceptor. Our data demonstrate that plants have a dGSH repair system that is directed to at least two cellular compartments via the use of alternative translation start sites.

Identification of a metabolic disposal route for the oncometabolite S-(2-succino)cysteine in Bacillus subtilis.

Cellular thiols such as cysteine spontaneously and readily react with the respiratory intermediate fumarate, resulting in the formation of stable S-(2-succino)-adducts. Fumarate-mediated succination of thiols increases in certain tumors and in response to glucotoxicity associated with diabetes. Therefore, S-(2-succino)-adducts such as S-(2-succino)cysteine (2SC) are considered oncometabolites and biomarkers for human disease. No disposal routes for S-(2-succino)-compounds have been reported prior to this study. Here, we show that Bacillus subtilis metabolizes 2SC to cysteine using a pathway encoded by the yxe operon. The first step is N-acetylation of 2SC followed by an oxygenation that we propose results in the release of oxaloacetate and N-acetylcysteine, which is deacetylated to give cysteine. Knockouts of the genes predicted to mediate each step in the pathway lose the ability to grow on 2SC as the sulfur source and accumulate the expected upstream metabolite(s). We further show that N-acetylation of 2SC relieves toxicity. This is the first demonstration of a metabolic disposal route for any S-(2-succino)-compound, paving the way toward the identification of corresponding pathways in other species.

Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase.

To synthesize the cofactor thiamin diphosphate (ThDP), plants must first hydrolyze thiamin monophosphate (ThMP) to thiamin, but dedicated enzymes for this hydrolysis step were unknown and widely doubted to exist. The classical thiamin-requiring th2-1 mutation in Arabidopsis thaliana was shown to reduce ThDP levels by half and to increase ThMP levels 5-fold, implying that the THIAMIN REQUIRING2 (TH2) gene product could be a dedicated ThMP phosphatase. Genomic and transcriptomic data indicated that TH2 corresponds to At5g32470, encoding a HAD (haloacid dehalogenase) family phosphatase fused to a TenA (thiamin salvage) family protein. Like the th2-1 mutant, an insertional mutant of At5g32470 accumulated ThMP, and the thiamin requirement of the th2-1 mutant was complemented by wild-type At5g32470 Complementation tests in Escherichia coli and enzyme assays with recombinant proteins confirmed that At5g32470 and its maize (Zea mays) orthologs GRMZM2G148896 and GRMZM2G078283 are ThMP-selective phosphatases whose activity resides in the HAD domain and that the At5g32470 TenA domain has the expected thiamin salvage activity. In vitro and in vivo experiments showed that alternative translation start sites direct the At5g32470 protein to the cytosol and potentially also to mitochondria. Our findings establish that plants have a dedicated ThMP phosphatase and indicate that modest (50%) ThDP depletion can produce severe deficiency symptoms.

Discovery of a widespread prokaryotic 5-oxoprolinase that was hiding in plain sight.

5-Oxoproline (OP) is well-known as an enzymatic intermediate in the eukaryotic γ-glutamyl cycle, but it is also an unavoidable damage product formed spontaneously from glutamine and other sources. Eukaryotes metabolize OP via an ATP-dependent 5-oxoprolinase; most prokaryotes lack homologs of this enzyme (and the γ-glutamyl cycle) but are predicted to have some way to dispose of OP if its spontaneous formation in vivo is significant. Comparative analysis of prokaryotic genomes showed that the gene encoding pyroglutamyl peptidase, which removes N-terminal OP residues, clusters in diverse genomes with genes specifying homologs of a fungal lactamase (renamed prokaryotic 5-oxoprolinase A, pxpA) and homologs of allophanate hydrolase subunits (renamed pxpB and pxpC). Inactivation of Bacillus subtilis pxpA, pxpB, or pxpC genes slowed growth, caused OP accumulation in cells and medium, and prevented use of OP as a nitrogen source. Assays of cell lysates showed that ATP-dependent 5-oxoprolinase activity disappeared when pxpA, pxpB, or pxpC was inactivated. 5-Oxoprolinase activity could be reconstituted in vitro by mixing recombinant B. subtilis PxpA, PxpB, and PxpC proteins. In addition, overexpressing Escherichia coli pxpABC genes in E. coli increased 5-oxoprolinase activity in lysates ≥1700-fold. This work shows that OP is a major universal metabolite damage product and that OP disposal systems are common in all domains of life. Furthermore, it illustrates how easily metabolite damage and damage-control systems can be overlooked, even for central metabolites in model organisms.

Mapping a kingdom-specific functional domain of squalene synthase.

Squalene synthase catalyzes the first committed step in sterol biosynthesis and consists of both an amino-terminal catalytic domain and a carboxy-terminal domain tethering the enzyme to the ER membrane. While the overall architecture of this enzyme is identical in eukaryotes, it was previously shown that plant and animal genes cannot complement a squalene synthase knockout mutation in yeast unless the carboxy-terminal domain is swapped for one of fungal origin. This implied a unique component of the fungal carboxy-terminal domain was responsible for the complementation phenotype. To identify this motif, we used Saccharomyces cerevisiae with a squalene synthase knockout mutation, and expressed intact and chimeric squalene synthases originating from fungi, plants, and animals. In contrast to previous observations, all enzymes tested could partially complement the knockout mutation when the genes were weakly expressed. However, when highly expressed, non-fungal squalene synthases could not complement the yeast mutation and instead led to the accumulation of a toxic intermediate(s) as defined by mutations of genes downstream in the ergosterol pathway. Restoration of the complete complementation phenotype was mapped to a 26-amino acid hinge region linking the catalytic and membrane-spanning domains specific to fungal squalene synthases. Over-expression of the C-terminal domain containing a hinge domain from fungi, not from animals or plants, led to growth inhibition of wild-type yeast. Because this hinge region is unique to and highly conserved within each kingdom of life, the data suggests that the hinge domain plays an essential functional role, such as assembly of ergosterol multi-enzyme complexes in fungi.

Arabidopsis and maize RidA proteins preempt reactive enamine/imine damage to branched-chain amino acid biosynthesis in plastids.

RidA (for Reactive Intermediate Deaminase A) proteins are ubiquitous, yet their function in eukaryotes is unclear. It is known that deleting Salmonella enterica ridA causes Ser sensitivity and that S. enterica RidA and its homologs from other organisms hydrolyze the enamine/imine intermediates that Thr dehydratase forms from Ser or Thr. In S. enterica, the Ser-derived enamine/imine inactivates a branched-chain aminotransferase; RidA prevents this damage. Arabidopsis thaliana and maize (Zea mays) have a RidA homolog that is predicted to be plastidial. Expression of either homolog complemented the Ser sensitivity of the S. enterica ridA mutant. The purified proteins hydrolyzed the enamines/imines formed by Thr dehydratase from Ser or Thr and protected the Arabidopsis plastidial branched-chain aminotransferase BCAT3 from inactivation by the Ser-derived enamine/imine. In vitro chloroplast import assays and in vivo localization of green fluorescent protein fusions showed that Arabidopsis RidA and Thr dehydratase are chloroplast targeted. Disrupting Arabidopsis RidA reduced root growth and raised the root and shoot levels of the branched-chain amino acid biosynthesis intermediate 2-oxobutanoate; Ser treatment exacerbated these effects in roots. Supplying Ile reversed the root growth defect. These results indicate that plastidial RidA proteins can preempt damage to BCAT3 and Ile biosynthesis by hydrolyzing the Ser-derived enamine/imine product of Thr dehydratase.

High-throughput comparison, functional annotation, and metabolic modeling of plant genomes using the PlantSEED resource.

The increasing number of sequenced plant genomes is placing new demands on the methods applied to analyze, annotate, and model these genomes. Today's annotation pipelines result in inconsistent gene assignments that complicate comparative analyses and prevent efficient construction of metabolic models. To overcome these problems, we have developed the PlantSEED, an integrated, metabolism-centric database to support subsystems-based annotation and metabolic model reconstruction for plant genomes. PlantSEED combines SEED subsystems technology, first developed for microbial genomes, with refined protein families and biochemical data to assign fully consistent functional annotations to orthologous genes, particularly those encoding primary metabolic pathways. Seamless integration with its parent, the prokaryotic SEED database, makes PlantSEED a unique environment for cross-kingdom comparative analysis of plant and bacterial genomes. The consistent annotations imposed by PlantSEED permit rapid reconstruction and modeling of primary metabolism for all plant genomes in the database. This feature opens the unique possibility of model-based assessment of the completeness and accuracy of gene annotation and thus allows computational identification of genes and pathways that are restricted to certain genomes or need better curation. We demonstrate the PlantSEED system by producing consistent annotations for 10 reference genomes. We also produce a functioning metabolic model for each genome, gapfilling to identify missing annotations and proposing gene candidates for missing annotations. Models are built around an extended biomass composition representing the most comprehensive published to date. To our knowledge, our models are the first to be published for seven of the genomes analyzed.

Systematic identification and analysis of frequent gene fusion events in metabolic pathways.

BACKGROUND: Gene fusions are the most powerful type of in silico-derived functional associations. However, many fusion compilations were made when <100 genomes were available, and algorithms for identifying fusions need updating to handle the current avalanche of sequenced genomes. The availability of a large fusion dataset would help probe functional associations and enable systematic analysis of where and why fusion events occur. RESULTS: Here we present a systematic analysis of fusions in prokaryotes. We manually generated two training sets: (i) 121 fusions in the model organism Escherichia coli; (ii) 131 fusions found in B vitamin metabolism. These sets were used to develop a fusion prediction algorithm that captured the training set fusions with only 7 % false negatives and 50 % false positives, a substantial improvement over existing approaches. This algorithm was then applied to identify 3.8 million potential fusions across 11,473 genomes. The results of the analysis are available in a searchable database at http://modelseed.org/projects/fusions/ . A functional analysis identified 3,000 reactions associated with frequent fusion events and revealed areas of metabolism where fusions are particularly prevalent. CONCLUSIONS: Customary definitions of fusions were shown to be ambiguous, and a stricter one was proposed. Exploring the genes participating in fusion events showed that they most commonly encode transporters, regulators, and metabolic enzymes. The major rationales for fusions between metabolic genes appear to be overcoming pathway bottlenecks, avoiding toxicity, controlling competing pathways, and facilitating expression and assembly of protein complexes. Finally, our fusion dataset provides powerful clues to decipher the biological activities of domains of unknown function.

'Nothing of chemistry disappears in biology': the Top 30 damage-prone endogenous metabolites.

Many common metabolites are intrinsically unstable and reactive, and hence prone to chemical (i.e. non-enzymatic) damage in vivo Although this fact is widely recognized, the purely chemical side-reactions of metabolic intermediates can be surprisingly hard to track down in the literature and are often treated in an unprioritized case-by-case way. Moreover, spontaneous chemical side-reactions tend to be overshadowed today by side-reactions mediated by promiscuous ('sloppy') enzymes even though chemical damage to metabolites may be even more prevalent than damage from enzyme sloppiness, has similar outcomes, and is held in check by similar biochemical repair or pre-emption mechanisms. To address these limitations and imbalances, here we draw together and systematically integrate information from the (bio)chemical literature, from cheminformatics, and from genome-scale metabolic models to objectively define a 'Top 30' list of damage-prone metabolites. A foundational part of this process was to derive general reaction rules for the damage chemistries involved. The criteria for a 'Top 30' metabolite included predicted chemical reactivity, essentiality, and occurrence in diverse organisms. We also explain how the damage chemistry reaction rules ('operators') are implemented in the Chemical-Damage-MINE (CD-MINE) database (minedatabase.mcs.anl.gov/#/top30) to provide a predictive tool for many additional potential metabolite damage products. Lastly, we illustrate how defining a 'Top 30' list can drive genomics-enabled discovery of the enzymes of previously unrecognized damage-control systems, and how applying chemical damage reaction rules can help identify previously unknown peaks in metabolomics profiles.

Proteins of Unknown Biochemical Function: A Persistent Problem and a Roadmap to Help Overcome It.

The number of sequenced genomes is rapidly increasing, but functional annotation of the genes in these genomes lags far behind. Even in Arabidopsis (Arabidopsis thaliana), only approximately 40% of enzyme- and transporter-encoding genes have credible functional annotations, and this number is even lower in nonmodel plants. Functional characterization of unknown genes is a challenge, but various databases (e.g. for protein localization and coexpression) can be mined to provide clues. If homologous microbial genes exist-and about one-half the genes encoding unknown enzymes and transporters in Arabidopsis have microbial homologs-cross-kingdom comparative genomics can powerfully complement plant-based data. Multiple lines of evidence can strengthen predictions and warrant experimental characterization. In some cases, relatively quick tests in genetically tractable microbes can determine whether a prediction merits biochemical validation, which is costly and demands specialized skills.

Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114 (Rid) protein family.

BACKGROUND: It is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5'-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions. RESULTS: Phylogenetic analysis divided the Rid family into a widely distributed, apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5'-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate. CONCLUSIONS: Like the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine oxidases as well as by pyridoxal 5'-phosphate enzymes. The RidA subfamily has an additional damage pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined. Finally, the Rid4 to Rid7 subfamilies appear not to hydrolyze imines and thus remain mysterious.

Structure-function mapping of key determinants for hydrocarbon biosynthesis by squalene and squalene synthase-like enzymes from the green alga Botryococcus braunii race B.

Squalene and botryococcene are branched-chain, triterpene compounds that arise from the head-to-head condensation of two molecules of farnesyl diphosphate to yield 1'-1 and 1'-3 linkages, respectively. The enzymes that catalyze their formation have attracted considerable interest from the medical field as potential drug targets and the renewable energy sector for metabolic engineering efforts. Recently, the enzymes responsible for botryococcene and squalene biosynthesis in the green alga Botryococcus braunii race B were characterized. To better understand how the specificity for the 1'-1 and 1'-3 linkages was controlled, we attempted to identify the functional residues and/or domains responsible for this step in the catalytic cascade. Existing crystal structures for the mammalian squalene synthase and Staphylococcus dehydrosqualene synthase enzymes were exploited to develop molecular models for the B. braunii botryococcene and squalene synthase enzymes. Residues within the active sites that could mediate catalytic specificity were identified, and reciprocal mutants were created in an attempt to interconvert the reaction product specificity of the enzymes. We report here the identification of several amino acid positions contributing to the rearrangement of the cyclopropyl intermediate to squalene, but these same positions do not appear to be sufficient to account for the cyclopropyl rearrangement to give botryococcene.

A universal metabolite repair enzyme removes a strong inhibitor of the TCA cycle.

A prevalent side-reaction of succinate dehydrogenase oxidizes malate to enol-oxaloacetate (OAA), a metabolically inactive form of OAA that is a strong inhibitor of succinate dehydrogenase. We purified from cow heart mitochondria an enzyme (OAT1) with OAA tautomerase (OAT) activity that converts enol-OAA to the physiological keto-OAA form, and determined that it belongs to the highly conserved and previously uncharacterized Fumarylacetoacetate_hydrolase_domain-containing protein family. From all three domains of life, heterologously expressed proteins were shown to have strong OAT activity, and ablating the OAT1 homolog caused significant growth defects. In Escherichia coli, expression of succinate dehydrogenase was necessary for OAT1-associated growth defects to occur, and ablating OAT1 caused a significant increase in acetate and other metabolites associated with anaerobic respiration. OAT1 increased the succinate dehydrogenase reaction rate by 35% in in vitro assays with physiological concentrations of both succinate and malate. Our results suggest that OAT1 is a universal metabolite repair enzyme that is required to maximize aerobic respiration efficiency by preventing succinate dehydrogenase inhibition.

Systems and strategies for plant protein expression.

At least a quarter of the protein-encoding genes in plant genomes are predicted to encode enzymes for which no physiological function is known. Determining functions for these uncharacterized enzymes is key to understanding plant metabolism. Functional characterization typically requires expression and purification of recombinant enzymes to be used in enzyme assays and/or for protein structure elucidation studies. Here, we describe several practical considerations used to improve the heterologous expression and purification of Arabidopsis thaliana and Zea mays NAD(P)HX dehydratase (NAXD) and NAD(P)HX epimerase (NAXE), two enzymes that are involved in repair of chemically damaged NAD(P)H cofactors. We provide protocols for transit peptide prediction and construct design, expression in Escherichia coli, and purification of NAXD and NAXE. Many of these strategies are generally applicable to the purification of any plant protein.

Genome Sequences of Two Pseudomonas Isolates That Can Use Metformin as the Sole Nitrogen Source.

Metformin is a major water pollutant globally. We report the complete genome sequences of two pseudomonads, Pseudomonas sp. strain KHPS1 and Pseudomonas hydrolytica strain KHPS2, isolated from wastewater treatment plant sludge, which can grow on metformin as the nitrogen source. Both isolates contained ~80-kb plasmids that may contain metformin breakdown genes.

Enzyme promiscuity, metabolite damage, and metabolite damage control systems of the tricarboxylic acid cycle.

Promiscuous enzymes and spontaneous chemical reactions can convert normal cellular metabolites into noncanonical or damaged metabolites. These damaged metabolites can be a useless drain on metabolism and may be inhibitory and/or reactive, sometimes leading to toxicity. Thus, mechanisms to prevent metabolite damage from occurring (metabolite damage preemption) or to convert damaged metabolites back to physiological forms (metabolite repair) are essential for sustained operation of metabolic networks. Some iconic examples of metabolite damage and its repair or preemption are associated with the tricarboxylic acid (TCA) cycle, and other metabolite damage control systems are likely to exist here due to the inherent promiscuity of TCA cycle enzymes and reactivity of TCA cycle intermediates. Here, we review known metabolite damage reactions and metabolite damage control systems associated with the TCA cycle. This includes a previously unrecognized metabolite damage control system - an oxaloacetate (OAA) enol-keto tautomerase activity that is 'built-in' to the TCA cycle. This activity is required to remove the highly inhibitory enol form of OAA and is likely to be critical for TCA cycle operation. By cataloging these instances, we show that metabolite damage and its repair or preemption is a prevalent feature of the TCA cycle and suggest many more metabolite damage control systems are likely to exist.