Metabolite repair, or metabolite damage control, has emerged as a fundamental pillar of intermediary metabolism alongside catalysis and regulation. Enzyme promiscuity and spontaneous chemical reactions inevitably generate abnormal metabolites that can interfere with classical metabolic processes. Dedicated metabolite repair enzymes prevent or reverse such damage, thereby preserving metabolic integrity. Defects in these systems define a growing class of inborn errors of metabolism, several of which are now clinically recognized and, in some cases, treatable. We summarize recent advances in the discovery and characterization of metabolite damage and repair systems in the tricarboxylic acid cycle, glycolysis, and other pathways, highlighting examples with established or potential links to human disease. We outline strategies for identifying additional metabolite repair defects and discuss diagnostic challenges, therapeutic perspectives, and connections between metabolite repair and aging. Understanding metabolite repair exposes the mechanisms that safeguard metabolism and opens new paths toward molecular diagnosis and targeted therapy.

Succination occurs when the TCA cycle intermediate fumarate reacts with cellular thiols, such as cysteine, yielding the damaged metabolite S-(2-succino)cysteine (2SC). Increased fumarate levels result in global succination of thiol-containing macromolecules and metabolites, which has been implicated in many human diseases. 2SC is a chemically stable molecule; however, enzymatic breakdown pathways have been identified in prokaryotes that involve N-acetylation of 2SC followed by a breakdown step that results in the release of the succino-moiety and N-acetylcysteine (NAC). NAC must be metabolized to cysteine to be assimilated, but enzymes catalyzing NAC deacetylation had hitherto not been thoroughly characterized. Here, we describe three enzymes in Bacillus subtilis, ScmP, YhaA, and YtnL, that all possess high NAC deacetylase activity in vitro. All three enzymes are metal-dependent hydrolases that are most active with cobalt and show remarkable specificity to NAC compared to structurally related acetylated small molecules. Growth assays demonstrated that these genes are functionally redundant in B. subtilis, and growth on NAC is only severely compromised when all three genes are knocked out of the genome. Together, our biochemical and genetic studies complete the functional characterization of the three-step 2SC degradation pathway in B. subtilis.

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.

A prevalent side-reaction of succinate dehydrogenase (SDH) oxidizes malate to enol-oxaloacetate (OAA), a metabolically inactive form of OAA that is a strong inhibitor of SDH. 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 SDH is necessary for OAT1-associated growth defects to occur, and ablating OAT1 causes a significant increase in acetate and other metabolites associated with anaerobic respiration. OAT1 increased the SDH reaction rate by 35% in in vitro assays with physiological concentrations of both succinate and malate. Our results indicate that OAT1 is a universal metabolite repair enzyme that is required to maximize aerobic respiration efficiency by preventing SDH inhibition.

Metformin is commonly used to lower blood glucose levels and is one of the most widely used pharmaceuticals worldwide. Typical doses are high (0.5-2.0 g day-1) and the majority travels through the digestive system unabsorbed and enters the wastewater system. Metformin is not removed by standard wastewater treatments and eventually enters freshwater systems, where it can form N-chloro-derivatives that are toxic to fish and human cells. Thus, metformin is one of the most prevalent anthropogenic pollutants worldwide and there has been considerable interest in finding ways to remove it. We recently isolated Pseudomonads capable of growing on metformin as the sole nitrogen source. We identified candidate genes involved in metformin breakdown through genomic analyses informed by feeding studies. One candidate, a pair of genes that are located on 80kb extra-genomic plasmids, was shown to encode a heteromeric Ni-dependent hydrolase that converts metformin to guanylurea and dimethylamine. Metforminase activity of these gene products is now well established as our results confirm three recently published independent studies. Our isolated Pseudomonads also grow on biguanide, suggesting the existence of an additional breakdown enzyme. Another candidate gene located on the 80kb plasmids was shown to encode an aminohydrolase that converts biguanide to guanylurea. Biguanide may arise through successive N-demethylations of metformin or come from other sources. Our results suggest that the recent evolution of metforminase and biguanide hydrolase enzymes allow Pseudomonads to convert either metformin or biguanide to guanylurea, which can be assimilated by existing pathways.

Methanogens are essential for the complete remineralization of organic matter in anoxic environments. Most cultured methanogens are hydrogenotrophic, using H 2 as an electron donor to reduce CO 2 to CH 4 , but in the absence of H 2 many can also use formate. Formate dehydrogenase (Fdh) is essential for formate oxidation, where it transfers electrons for reduction of coenzyme F 420 or to a flavin-based electron bifurcating reaction catalyzed by heterodisulfide reductase (Hdr), the terminal reaction of methanogenesis. How these competing reactions are coordinated is unknown. Furthermore, methanogens that use formate encode at least two isoforms of Fdh in their genomes, but how these different isoforms participate in methanogenesis is also 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 F 420 at similar rates. F 420 reduction preceded flavin-based electron bifurcation activity for both enzymes. In a Δ fdh1 mutant background, a suppressor mutation was required for Fdh2 activity. Genome sequencing revealed that this mutation resulted in loss of a specific molybdopterin transferase ( moeA ), allowing for Fdh2-dependent growth. This suggests that both isoforms of Fdh are functionally redundant, but their activities in vivo may be limited by gene regulation under different growth conditions. Together these results expand our understanding of formate oxidation and the role of Fdh in methanogenesis.

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.

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.

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.

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 2SNAC. YxeK catalyzes the oxygenation of 2SNAC, resulting in its breakdown to oxaloacetate and N -acetylcysteine (NAC), 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 NAC, 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

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.

Phosphorylation is a reversible posttranslational modification that modulates the activities of numerous proteins.In plants,it is known that several mitochondrial proteins can be phosphory-lated,including enzymes of the tricarboxylic acid (TCA) cycle.The kinases and phosphorylases that mediate reversible phos-phorylation of TCA cycle enzymes remain largely elusive,as is the impact of protein phosphorylation on the TCA cycle.In this issue of Molecular Plant,Zhang et al.(2021) demonstrate that two phosphatases play important roles in controlling the phosphorylation status of TCA cycle enzymes,which modulates their activities and influences carbon flux through the TCA 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.

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 gamma-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 feast two cellular compartments via the use of alternative translation start sites.

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.

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NAD(P)H-hydrate epimerase (EC 5.1.99.6) is known to help repair NAD(P)H hydrates (NAD(P)HX), which are damage products existing as R and S epimers. The S epimer is reconverted to NAD(P)H by a dehydratase; the epimerase facilitates epimer interconversion. Epimerase deficiency in humans causes a lethal disorder attributed to NADHX accumulation. However, bioinformatic evidence suggest caution about this attribution by predicting that the epimerase has a second function connected to vitamin B6 (pyridoxal 5′-phosphate and related compounds). Specifically, (i) the epimerase is fused to a B6 salvage enzyme in plants, (ii) epimerase genes cluster on the chromosome with B6-related genes in bacteria, and (iii) epimerase and B6-related genes are coexpressed in yeast and Arabidopsis. The predicted second function was explored in Escherichia coli, whose epimerase and dehydratase are fused and encoded by yjeF. The putative NAD(P)HX epimerase active site has a conserved lysine residue (K192 in E. coli YjeF). Changing this residue to alanine cut in vitro epimerase activity by ≥95% but did not affect dehydratase activity. Mutant cells carrying the K192A mutation had essentially normal NAD(P)HX dehydratase activity and NAD(P)HX levels, showing that the mutation had little impact on NAD(P)HX repair in vivo. However, these cells showed metabolome changes, particularly in amino acids, which exceeded those in cells lacking the entire yjeF gene. The K192A mutant cells also had reduced levels of ‘free’ (i.e. weakly bound or unbound) pyridoxal 5'-phosphate. These results provide circumstantial evidence that the epimerase has a metabolic function beyond NAD(P)HX repair and that this function involves vitamin B6.

Thiamin is essential for plant growth but is short-lived in vivo and energetically very costly to produce a combination that makes thiamin biosynthesis a prime target for improvement by redesign. Thiamin consists of thiazole and pyrimidine moieties. Its high biosynthetic cost stems from use of the suicide enzyme THI4 to form the thiazole and the near-suicide enzyme THIC to form the pyrimidine. These energetic costs lower biomass yield potential and are likely compounded by environmental stresses that destroy thiamin and hence increase the rate at which it must be made. The energy costs could be slashed by refactoring the thiamin biosynthesis pathway to eliminate the suicidal THI4 and THIC reactions. To substantiate this design concept, we first document the energetic costs of the THI4 and THIC steps in the pathway and explain how cutting these costs could substantially increase crop biomass and grain yields. We then show that a refactored pathway must produce thiamin itself rather than a stripped-down analog because the thiamin molecule cannot be simplified without losing biological activity. Lastly, we consider possible energy-efficient alternatives to the inefficient natural THI4- and THIC-mediated steps.