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.

Severe NAD(P)HX Dehydratase (NAXD) Neurometabolic Syndrome May Present in Adulthood after Mild Head Trauma.

We have previously reported that pathogenic variants in a key metabolite repair enzyme NAXD cause a lethal neurodegenerative condition triggered by episodes of fever in young children. However, the clinical and genetic spectrum of NAXD deficiency is broadening as our understanding of the disease expands and as more cases are identified. Here, we report the oldest known individual succumbing to NAXD-related neurometabolic crisis, at 32 years of age. The clinical deterioration and demise of this individual were likely triggered by mild head trauma. This patient had a novel homozygous NAXD variant [NM_001242882.1:c.441+3A>G:p.?] that induces the mis-splicing of the majority of NAXD transcripts, leaving only trace levels of canonically spliced NAXD mRNA, and protein levels below the detection threshold by proteomic analysis. Accumulation of damaged NADH, the substrate of NAXD, could be detected in the fibroblasts of the patient. In agreement with prior anecdotal reports in paediatric patients, niacin-based treatment also partly alleviated some clinical symptoms in this adult patient. The present study extends our understanding of NAXD deficiency by uncovering shared mitochondrial proteomic signatures between the adult and our previously reported paediatric NAXD cases, with reduced levels of respiratory complexes I and IV as well as the mitoribosome, and the upregulation of mitochondrial apoptotic pathways. Importantly, we highlight that head trauma in adults, in addition to paediatric fever or illness, may precipitate neurometabolic crises associated with pathogenic NAXD variants.

The Limits of Enzyme Specificity and the Evolution of Metabolism.

The substrate specificity of enzymes is bound to be imperfect, because of unavoidable physicochemical limits. In extant metabolic enzymes, furthermore, such limits are seldom approached, suggesting that the degree of specificity of these enzymes, on average, is much lower than could be attained. During biological evolution, the activity of a single enzyme with available alternative substrates may be preserved to a significant or even substantial level for different reasons - for example when the alternative reaction contributes to fitness, or when its undesirable products are nevertheless dispatched by metabolite repair enzymes. In turn, the widespread occurrence of promiscuous reactions is a consistent source of metabolic 'messiness', from which both liabilities and opportunities ensue in the evolution of metabolic systems.

Non-canonical metabolic pathways in the malaria parasite detected by isotope-tracing metabolomics.

The malaria parasite, Plasmodium falciparum, proliferates rapidly in human erythrocytes by actively scavenging multiple carbon sources and essential nutrients from its host cell. However, a global overview of the metabolic capacity of intraerythrocytic stages is missing. Using multiplex 13 C-labelling coupled with untargeted mass spectrometry and unsupervised isotopologue grouping, we have generated a draft metabolome of P. falciparum and its host erythrocyte consisting of 911 and 577 metabolites, respectively, corresponding to 41% of metabolites and over 70% of the metabolic reaction predicted from the parasite genome. An additional 89 metabolites and 92 reactions were identified that were not predicted from genomic reconstructions, with the largest group being associated with metabolite damage-repair systems. Validation of the draft metabolome revealed four previously uncharacterised enzymes which impact isoprenoid biosynthesis, lipid homeostasis and mitochondrial metabolism and are necessary for parasite development and proliferation. This study defines the metabolic fate of multiple carbon sources in P. falciparum, and highlights the activity of metabolite repair pathways in these rapidly growing parasite stages, opening new avenues for drug discovery.

Clinical and biochemical distinctions for a metabolite repair disorder caused by NAXD or NAXE deficiency.

The central cofactors NAD(P)H are prone to damage by hydration, resulting in formation of redox-inactive derivatives designated NAD(P)HX. The highly conserved enzymes NAD(P)HX dehydratase (NAXD) and NAD(P)HX epimerase (NAXE) function to repair intracellular NAD(P)HX. Recently, pathogenic variants in both the NAXD and NAXE genes were associated with rapid deterioration and death after an otherwise trivial fever, infection, or illness in young patients. As more patients are identified, distinct clinical features are emerging depending on the location of the pathogenic variant. In this review, we carefully catalogued the clinical features of all published NAXD deficiency patients and found distinct patterns in clinical presentations depending on which subcellular compartment is affected by the enzymatic deficiency. Exon 1 of NAXD contains a mitochondrial propeptide, and a unique cytosolic isoform is initiated from an alternative start codon in exon 2. NAXD deficiency patients with variants that affect both the cytosolic and mitochondrial isoforms present with neurological defects, seizures and skin lesions. Interestingly, patients with NAXD variants exclusively affecting the mitochondrial isoform present with myopathy, moderate neuropathy and a cardiac presentation, without the characteristic skin lesions, seizures or neurological degeneration. This suggests that cytosolic NAD(P)HX repair may protect from neurological damage, whereas muscle fibres may be more sensitive to mitochondrial NAD(P)HX damage. A deeper understanding of the clinical phenotype may facilitate rapid identification of new cases and allow earlier therapeutic intervention. Niacin-based therapies are promising, but advances in disease modelling for both NAXD and NAXE deficiency may identify more specific compounds as targeted treatments. In this review, we found distinct patterns in the clinical presentations of NAXD deficiency patients based on the location of the pathogenic variant, which determines the subcellular compartment that is affected by the enzymatic deficiency.

Failure to eliminate a phosphorylated glucose analog leads to neutropenia in patients with G6PT and G6PC3 deficiency.

Neutropenia represents an important problem in patients with genetic deficiency in either the glucose-6-phosphate transporter of the endoplasmic reticulum (G6PT/SLC37A4) or G6PC3, an endoplasmic reticulum phosphatase homologous to glucose-6-phosphatase. While affected granulocytes show reduced glucose utilization, the underlying mechanism is unknown and causal therapies are lacking. Using a combination of enzymological, cell-culture, and in vivo approaches, we demonstrate that G6PT and G6PC3 collaborate to destroy 1,5-anhydroglucitol-6-phosphate (1,5AG6P), a close structural analog of glucose-6-phosphate and an inhibitor of low-KM hexokinases, which catalyze the first step in glycolysis in most tissues. We show that 1,5AG6P is made by phosphorylation of 1,5-anhydroglucitol, a compound normally present in human plasma, by side activities of ADP-glucokinase and low-KM hexokinases. Granulocytes from patients deficient in G6PC3 or G6PT accumulate 1,5AG6P to concentrations (∼3 mM) that strongly inhibit hexokinase activity. In a model of G6PC3-deficient mouse neutrophils, physiological concentrations of 1,5-anhydroglucitol caused massive accumulation of 1,5AG6P, a decrease in glucose utilization, and cell death. Treating G6PC3-deficient mice with an inhibitor of the kidney glucose transporter SGLT2 to lower their blood level of 1,5-anhydroglucitol restored a normal neutrophil count, while administration of 1,5-anhydroglucitol had the opposite effect. In conclusion, we show that the neutropenia in patients with G6PC3 or G6PT mutations is a metabolite-repair deficiency, caused by a failure to eliminate the nonclassical metabolite 1,5AG6P.

A Guardian Angel Phosphatase for Mainline Carbon Metabolism.

It is increasingly clear that many metabolic enzymes mistakenly form minor but toxic side-products that must be eliminated to maintain normal fluxes. Collard et al. show that this is true of two iconic glycolytic enzymes, and that a hitherto somewhat mysterious phosphatase rescues central carbon metabolism from their mistakes.

Human ARMT1 structure and substrate specificity indicates that it is a DUF89 family damage-control phosphatase.

Metabolite damage control is a critical but poorly defined aspect of cellular biochemistry, which likely involves many of the so far functionally uncharacterized protein domain (domains of unknown function; DUFs). We have determined the crystal structure of the human DUF89 protein product of the C6ORF211 gene to 1.85 Å. The crystal structure shows that the protein contains a core α-ß-α fold with an active site-bound metal ion and α-helical bundle N-terminal cap, which are both conserved features of subfamily III DUF89 domains. The biochemical activities of the human protein are conserved with those of a previously characterized budding yeast homolog, where an in vitro phosphatase activity is supported by divalent cations that include Co2+, Ni2+, Mn2+ or Mg2+. Full steady-state kinetics parameters of human DUF89 using a standard PNPP phosphatase assay revealed a six times higher catalytic efficiency in presence of Co2+ compared to Mg2+. The human enzyme targets a number of phosphate substrates similar to the budding yeast homolog, while it lacks a previously indicated methyltransferase activity. The highest activity on substrate was observed with fructose-1-phosphate, a potent glycating agent, and thus human DUF89 phosphatase activity may also play a role in limiting the buildup of phospho-glycan species and their related damaged metabolites.

Hypothesis: A Novel Neuroprotective Role for Glucose-6-phosphatase (G6PC3) in Brain-To Maintain Energy-Dependent Functions Including Cognitive Processes.

The isoform of glucose-6-phosphatase in liver, G6PC1, has a major role in whole-body glucose homeostasis, whereas G6PC3 is widely distributed among organs but has poorly-understood functions. A recent, elegant analysis of neutrophil dysfunction in G6PC3-deficient patients revealed G6PC3 is a neutrophil metabolite repair enzyme that hydrolyzes 1,5-anhydroglucitol-6-phosphate, a toxic metabolite derived from a glucose analog present in food. These patients exhibit a spectrum of phenotypic characteristics and some have learning disabilities, revealing a potential linkage between cognitive processes and G6PC3 activity. Previously-debated and discounted functions for brain G6PC3 include causing an ATP-consuming futile cycle that interferes with metabolic brain imaging assays and a nutritional role involving astrocyte-neuron glucose-lactate trafficking. Detailed analysis of the anhydroglucitol literature reveals that it competes with glucose for transport into brain, is present in human cerebrospinal fluid, and is phosphorylated by hexokinase. Anhydroglucitol-6-phosphate is present in rodent brain and other organs where its accumulation can inhibit hexokinase by competition with ATP. Calculated hexokinase inhibition indicates that energetics of brain and erythrocytes would be more adversely affected by anhydroglucitol-6-phosphate accumulation than heart. These findings strongly support the paradigm-shifting hypothesis that brain G6PC3 removes a toxic metabolite, thereby maintaining brain glucose metabolism- and ATP-dependent functions, including cognitive processes.

Inborn errors of metabolite repair.

It is traditionally assumed that enzymes of intermediary metabolism are extremely specific and that this is sufficient to prevent the production of useless and/or toxic side-products. Recent work indicates that this statement is not entirely correct. In reality, enzymes are not strictly specific, they often display weak side activities on intracellular metabolites (substrate promiscuity) that resemble their physiological substrate or slowly catalyse abnormal reactions on their physiological substrate (catalytic promiscuity). They thereby produce non-classical metabolites that are not efficiently metabolised by conventional enzymes. In an increasing number of cases, metabolite repair enzymes are being discovered that serve to eliminate these non-classical metabolites and prevent their accumulation. Metabolite repair enzymes also eliminate non-classical metabolites that are formed through spontaneous (ie, not enzyme-catalysed) reactions. Importantly, genetic deficiencies in several metabolite repair enzymes lead to 'inborn errors of metabolite repair', such as L-2-hydroxyglutaric aciduria, D-2-hydroxyglutaric aciduria, 'ubiquitous glucose-6-phosphatase' (G6PC3) deficiency, the neutropenia present in Glycogen Storage Disease type Ib or defects in the enzymes that repair the hydrated forms of NADH or NADPH. Metabolite repair defects may be difficult to identify as such, because the mutated enzymes are non-classical enzymes that act on non-classical metabolites, which in some cases accumulate only inside the cells, and at rather low, yet toxic, concentrations. It is therefore likely that many additional metabolite repair enzymes remain to be discovered and that many diseases of metabolite repair still await elucidation.

Plant nitrilase: a new job for an old enzyme.

Nitrilases are versatile enzymes that hydrolyze nitriles to carboxylic acids and ammonia, but many members of this family lack defined biological functions. In plants, nitrilases have been associated with detoxification of cyanide-containing compounds and auxin biosynthesis; however, recent work suggests that the chemical versatility of these proteins contributes to metabolite repair. In this issue of the Biochemical Journal, Niehaus et al. demonstrate that the Nit1 nitrilase from Arabidopsis thaliana functions as a metabolite repair enzyme that removes deaminated glutathione from the cytoplasm and plastids.

Pyridoxamine-phosphate oxidases and pyridoxamine-phosphate oxidase-related proteins catalyze the oxidation of 6-NAD(P)H to NAD(P).

6-NADH and 6-NADPH are strong inhibitors of several dehydrogenases that may form spontaneously from NAD(P)H. They are known to be oxidized to NAD(P)+ by mammalian renalase, an FAD-linked enzyme mainly present in heart and kidney, and by related bacterial enzymes. We partially purified an enzyme oxidizing 6-NADPH from rat liver, and, surprisingly, identified it as pyridoxamine-phosphate oxidase (PNPO). This was confirmed by the finding that recombinant mouse PNPO oxidized 6-NADH and 6-NADPH with catalytic efficiencies comparable to those observed with pyridoxine- and pyridoxamine-5'-phosphate. PNPOs from Escherichia coli, Saccharomyces cerevisiae and Arabidopsis thaliana also displayed 6-NAD(P)H oxidase activity, indicating that this 'side-activity' is conserved. Remarkably, 'pyridoxamine-phosphate oxidase-related proteins' (PNPO-RP) from Nostoc punctiforme, A. thaliana and the yeast S. cerevisiae (Ygr017w) were not detectably active on pyridox(am)ine-5'-P, but oxidized 6-NADH, 6-NADPH and 2-NADH suggesting that this may be their main catalytic function. Their specificity profiles were therefore similar to that of renalase. Inactivation of renalase and of PNPO in mammalian cells and of Ygr017w in yeasts led to the accumulation of a reduced form of 6-NADH, tentatively identified as 4,5,6-NADH3, which can also be produced in vitro by reduction of 6-NADH by glyceraldehyde-3-phosphate dehydrogenase or glucose-6-phosphate dehydrogenase. As 4,5,6-NADH3 is not a substrate for renalase, PNPO or PNPO-RP, its accumulation presumably reflects the block in the oxidation of 6-NADH. These findings indicate that two different classes of enzymes using either FAD (renalase) or FMN (PNPOs and PNPO-RPs) as a cofactor play an as yet unsuspected role in removing damaged forms of NAD(P).

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.

The synthesis of branched-chain fatty acids is limited by enzymatic decarboxylation of ethyl- and methylmalonyl-CoA.

Most fatty acids (FAs) are straight chains and are synthesized by fatty acid synthase (FASN) using acetyl-CoA and malonyl-CoA units. Yet, FASN is known to be promiscuous as it may use methylmalonyl-CoA instead of malonyl-CoA and thereby introduce methyl-branches. We have recently found that the cytosolic enzyme ECHDC1 degrades ethylmalonyl-CoA and methylmalonyl-CoA, which presumably result from promiscuous reactions catalyzed by acetyl-CoA carboxylase on butyryl- and propionyl-CoA. Here, we tested the hypothesis that ECHDC1 is a metabolite repair enzyme that serves to prevent the formation of methyl- or ethyl-branched FAs by FASN. Using the purified enzyme, we found that FASN can incorporate not only methylmalonyl-CoA but also ethylmalonyl-CoA, producing methyl- or ethyl-branched FAs. Using a combination of gas-chromatography and liquid chromatography coupled to mass spectrometry, we observed that inactivation of ECHDC1 in adipocytes led to an increase in several methyl-branched FAs (present in different lipid classes), while its overexpression reduced them below wild-type levels. In contrast, the formation of ethyl-branched FAs was observed almost exclusively in ECHDC1 knockout cells, indicating that ECHDC1 and the low activity of FASN toward ethylmalonyl-CoA efficiently prevent their formation. We conclude that ECHDC1 performs a typical metabolite repair function by destroying methyl- and ethylmalonyl-CoA. This reduces the formation of methyl-branched FAs and prevents the formation of ethyl-branched FAs by FASN. The identification of ECHDC1 as a key modulator of the abundance of methyl-branched FAs opens the way to investigate their function.

Nit1 is a metabolite repair enzyme that hydrolyzes deaminated glutathione.

The mammalian gene Nit1 (nitrilase-like protein 1) encodes a protein that is highly conserved in eukaryotes and is thought to act as a tumor suppressor. Despite being ∼35% sequence identical to ω-amidase (Nit2), the Nit1 protein does not hydrolyze efficiently α-ketoglutaramate (a known physiological substrate of Nit2), and its actual enzymatic function has so far remained a puzzle. In the present study, we demonstrate that both the mammalian Nit1 and its yeast ortholog are amidases highly active toward deaminated glutathione (dGSH; i.e., a form of glutathione in which the free amino group has been replaced by a carbonyl group). We further show that Nit1-KO mutants of both human and yeast cells accumulate dGSH and the same compound is excreted in large amounts in the urine of Nit1-KO mice. Finally, we show that several mammalian aminotransferases (transaminases), both cytosolic and mitochondrial, can form dGSH via a common (if slow) side-reaction and provide indirect evidence that transaminases are mainly responsible for dGSH formation in cultured mammalian cells. Altogether, these findings delineate a typical instance of metabolite repair, whereby the promiscuous activity of some abundant enzymes of primary metabolism leads to the formation of a useless and potentially harmful compound, which needs a suitable "repair enzyme" to be destroyed or reconverted into a useful metabolite. The need for a dGSH repair reaction does not appear to be limited to eukaryotes: We demonstrate that Nit1 homologs acting as excellent dGSH amidases also occur in Escherichia coli and other glutathione-producing bacteria.

NAXE Mutations Disrupt the Cellular NAD(P)HX Repair System and Cause a Lethal Neurometabolic Disorder of Early Childhood.

To safeguard the cell from the accumulation of potentially harmful metabolic intermediates, specific repair mechanisms have evolved. APOA1BP, now renamed NAXE, encodes an epimerase essential in the cellular metabolite repair for NADHX and NADPHX. The enzyme catalyzes the epimerization of NAD(P)HX, thereby avoiding the accumulation of toxic metabolites. The clinical importance of the NAD(P)HX repair system has been unknown. Exome sequencing revealed pathogenic biallelic mutations in NAXE in children from four families with (sub-) acute-onset ataxia, cerebellar edema, spinal myelopathy, and skin lesions. Lactate was elevated in cerebrospinal fluid of all affected individuals. Disease onset was during the second year of life and clinical signs as well as episodes of deterioration were triggered by febrile infections. Disease course was rapidly progressive, leading to coma, global brain atrophy, and finally to death in all affected individuals. NAXE levels were undetectable in fibroblasts from affected individuals of two families. In these fibroblasts we measured highly elevated concentrations of the toxic metabolite cyclic-NADHX, confirming a deficiency of the mitochondrial NAD(P)HX repair system. Finally, NAD or nicotinic acid (vitamin B3) supplementation might have therapeutic implications for this fatal disorder.

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.

Metabolite damage and repair in metabolic engineering design.

The necessarily sharp focus of metabolic engineering and metabolic synthetic biology on pathways and their fluxes has tended to divert attention from the damaging enzymatic and chemical side-reactions that pathway metabolites can undergo. Although historically overlooked and underappreciated, such metabolite damage reactions are now known to occur throughout metabolism and to generate (formerly enigmatic) peaks detected in metabolomics datasets. It is also now known that metabolite damage is often countered by dedicated repair enzymes that undo or prevent it. Metabolite damage and repair are highly relevant to engineered pathway design: metabolite damage reactions can reduce flux rates and product yields, and repair enzymes can provide robust, host-independent solutions. Herein, after introducing the core principles of metabolite damage and repair, we use case histories to document how damage and repair processes affect efficient operation of engineered pathways - particularly those that are heterologous, non-natural, or cell-free. We then review how metabolite damage reactions can be predicted, how repair reactions can be prospected, and how metabolite damage and repair can be built into genome-scale metabolic models. Lastly, we propose a versatile 'plug and play' set of well-characterized metabolite repair enzymes to solve metabolite damage problems known or likely to occur in metabolic engineering and synthetic biology projects.

Crystal structure of the homocysteine methyltransferase MmuM from Escherichia coli.

Homocysteine S-methyltransferases (HMTs, EC 2.1.1.0) catalyse the conversion of homocysteine to methionine using S-methylmethionine or S-adenosylmethionine as the methyl donor. HMTs play an important role in methionine biosynthesis and are widely distributed among micro-organisms, plants and animals. Additionally, HMTs play a role in metabolite repair of S-adenosylmethionine by removing an inactive diastereomer from the pool. The mmuM gene product from Escherichia coli is an archetypal HMT family protein and contains a predicted zinc-binding motif in the enzyme active site. In the present study, we demonstrate X-ray structures for MmuM in oxidized, apo and metallated forms, representing the first such structures for any member of the HMT family. The structures reveal a metal/substrate-binding pocket distinct from those in related enzymes. The presented structure analysis and modelling of co-substrate interactions provide valuable insight into the function of MmuM in both methionine biosynthesis and cofactor repair.

A pathway for repair of NAD(P)H in plants.

Unwanted enzyme side reactions and spontaneous decomposition of metabolites can lead to a build-up of compounds that compete with natural enzyme substrates and must be dealt with for efficient metabolism. It has recently been realized that there are enzymes that process such compounds, formulating the concept of metabolite repair. NADH and NADPH are vital cellular redox cofactors but can form non-functional hydrates (named NAD(P)HX) spontaneously or enzymatically that compete with enzymes dependent on NAD(P)H, impairing normal enzyme function. Here we report on the functional characterization of components of a potential NAD(P)H repair pathway in plants comprising a stereospecific dehydratase (NNRD) and an epimerase (NNRE), the latter being fused to a vitamin B6 salvage enzyme. Through the use of the recombinant proteins, we show that the ATP-dependent NNRD and NNRE act concomitantly to restore NAD(P)HX to NAD(P)H. NNRD behaves as a tetramer and NNRE as a dimer, but the proteins do not physically interact. In vivo fluorescence analysis demonstrates that the proteins are localized to mitochondria and/or plastids, implicating these as the key organelles where this repair is required. Expression analysis indicates that whereas NNRE is present ubiquitously, NNRD is restricted to seeds but appears to be dispensable during the normal Arabidopsis life cycle.