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highly active toward deaminated glutathione (dGSH; i.e., a form of glutathione in which the free amino group has been replaced by a carbonyl group).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.

Canadian regulatory aspects of gene editing technologies.

The development of gene editing techniques, capable of producing plants and animals with new and improved traits, is revolutionizing the world of plant and animal breeding and rapidly advancing to commercial reality. However, from a regulatory standpoint the Government of Canada views gene editing as another tool that will join current methods used to develop desirable traits in plants and animals. This is because Canada focusses on the potential risk resulting from the novelty of the trait, or plant or animal product entering the Canadian environment or market place, rather than the process or method by which it was created. The Canadian Food Inspection Agency is responsible for the regulation of the environmental release of plants with novel traits, and novel livestock feeds, while Health Canada is responsible for the regulation of novel foods. Environment and Climate Change Canada, in partnership with Health Canada, regulates modified animals for entry into the environment. In all cases, these novel products may be the result of conventional breeding, mutagenesis, recombinant DNA techniques or other methods of plant or animal breeding such as gene editing. This novelty approach allows the Canadian regulatory system to efficiently adjust to any new developments in the science of plant and animal breeding and allows for risk-appropriate regulatory decisions. This approach encourages innovation while maintaining science-based regulatory expertise. Canadian regulators work cooperatively with proponents to determine if their gene editing-derived product meets the definition of a novel product, and whether it would be subject to a pre-market assessment. Therefore, Canada's existing regulatory system is well positioned to accommodate any new innovations or technologies in plant or animal breeding, including gene editing.

Confronting the catalytic dark matter encoded by sequenced genomes.

The post-genomic era has provided researchers with a deluge of protein sequences. However, a significant fraction of the proteins encoded by sequenced genomes remains without an identified function. Here, we aim at determining how many enzymes of uncertain or unknown function are still present in the Saccharomyces cerevisiae and human proteomes. Using information available in the Swiss-Prot, BRENDA and KEGG databases in combination with a Hidden Markov Model-based method, we estimate that >600 yeast and 2000 human proteins (>30% of their proteins of unknown function) are enzymes whose precise function(s) remain(s) to be determined. This illustrates the impressive scale of the 'unknown enzyme problem'. We extensively review classical biochemical as well as more recent systematic experimental and computational approaches that can be used to support enzyme function discovery research. Finally, we discuss the possible roles of the elusive catalysts in light of recent developments in the fields of enzymology and metabolism as well as the significance of the unknown enzyme problem in the context of metabolic modeling, metabolic engineering and rare disease research.

Identification of mitochondrial coenzyme a transporters from maize and Arabidopsis.

Plants make coenzyme A (CoA) in the cytoplasm but use it for reactions in mitochondria, chloroplasts, and peroxisomes, implying that these organelles have CoA transporters. A plant peroxisomal CoA transporter is already known, but plant mitochondrial or chloroplastic CoA transporters are not. Mitochondrial CoA transporters belonging to the mitochondrial carrier family, however, have been identified in yeast (Saccharomyces cerevisiae; Leu-5p) and mammals (SLC25A42). Comparative genomic analysis indicated that angiosperms have two distinct homologs of these mitochondrial CoA transporters, whereas nonflowering plants have only one. The homologs from maize (Zea mays; GRMZM2G161299 and GRMZM2G420119) and Arabidopsis (Arabidopsis thaliana; At1g14560 and At4g26180) all complemented the growth defect of the yeast leu5Δ mitochondrial CoA carrier mutant and substantially restored its mitochondrial CoA level, confirming that these proteins have CoA transport activity. Dual-import assays with purified pea (Pisum sativum) mitochondria and chloroplasts, and subcellular localization of green fluorescent protein fusions in transiently transformed tobacco (Nicotiana tabacum) Bright Yellow-2 cells, showed that the maize and Arabidopsis proteins are targeted to mitochondria. Consistent with the ubiquitous importance of CoA, the maize and Arabidopsis mitochondrial CoA transporter genes are expressed at similar levels throughout the plant. These data show that representatives of both monocotyledons and eudicotyledons have twin, mitochondrially located mitochondrial carrier family carriers for CoA. The highly conserved nature of these carriers makes possible their reliable annotation in other angiosperm genomes.

Identification and characterization of the missing pyrimidine reductase in the plant riboflavin biosynthesis pathway.

Riboflavin (vitamin B2) is the precursor of the flavin coenzymes flavin mononucleotide and flavin adenine dinucleotide. In Escherichia coli and other bacteria, sequential deamination and reduction steps in riboflavin biosynthesis are catalyzed by RibD, a bifunctional protein with distinct pyrimidine deaminase and reductase domains. Plants have two diverged RibD homologs, PyrD and PyrR; PyrR proteins have an extra carboxyl-terminal domain (COG3236) of unknown function. Arabidopsis (Arabidopsis thaliana) PyrD (encoded by At4g20960) is known to be a monofunctional pyrimidine deaminase, but no pyrimidine reductase has been identified. Bioinformatic analyses indicated that plant PyrR proteins have a catalytically competent reductase domain but lack essential zinc-binding residues in the deaminase domain, and that the Arabidopsis PyrR gene (At3g47390) is coexpressed with riboflavin synthesis genes. These observations imply that PyrR is a pyrimidine reductase without deaminase activity. Consistent with this inference, Arabidopsis or maize (Zea mays) PyrR (At3g47390 or GRMZM2G090068) restored riboflavin prototrophy to an E. coli ribD deletant strain when coexpressed with the corresponding PyrD protein (At4g20960 or GRMZM2G320099) but not when expressed alone; the COG3236 domain was unnecessary for complementing activity. Furthermore, recombinant maize PyrR mediated NAD(P)H-dependent pyrimidine reduction in vitro. Import assays with pea (Pisum sativum) chloroplasts showed that PyrR and PyrD are taken up and proteolytically processed. Ablation of the maize PyrR gene caused early seed lethality. These data argue that PyrR is the missing plant pyrimidine reductase, that it is plastid localized, and that it is essential. The role of the COG3236 domain remains mysterious; no evidence was obtained for the possibility that it catalyzes the dephosphorylation that follows pyrimidine reduction.

Evidence that the folate-dependent proteins YgfZ and MnmEG have opposing effects on growth and on activity of the iron-sulfur enzyme MiaB.

The folate-dependent protein YgfZ of Escherichia coli participates in the synthesis and repair of iron-sulfur (Fe-S) clusters; it belongs to a family of enzymes that use folate to capture formaldehyde units. Ablation of ygfZ is known to reduce growth, to increase sensitivity to oxidative stress, and to lower the activities of MiaB and other Fe-S enzymes. It has been reported that the growth phenotype can be suppressed by disrupting the tRNA modification gene mnmE. We first confirmed the latter observation using deletions in a simpler, more defined genetic background. We then showed that deleting mnmE substantially restores MiaB activity in ygfZ deletant cells and that overexpressing MnmE with its partner MnmG exacerbates the growth and MiaB activity phenotypes of the ygfZ deletant. MnmE, with MnmG, normally mediates a folate-dependent transfer of a formaldehyde unit to tRNA, and the MnmEG-mediated effects on the phenotypes of the ΔygfZ mutant apparently require folate, as evidenced by the effect of eliminating all folates by deleting folE. The expression of YgfZ was unaffected by deleting mnmE or overexpressing MnmEG or by folate status. Since formaldehyde transfer is a potential link between MnmEG and YgfZ, we inactivated formaldehyde detoxification by deleting frmA. This deletion had little effect on growth or MiaB activity in the ΔygfZ strain in the presence of formaldehyde, making it unlikely that formaldehyde alone connects the actions of MnmEG and YgfZ. A more plausible explanation is that MnmEG erroneously transfers a folate-bound formaldehyde unit to MiaB and that YgfZ reverses this.

Mitochondrial and plastidial COG0354 proteins have folate-dependent functions in iron-sulphur cluster metabolism.

COG0354 proteins have been implicated in synthesis or repair of iron/sulfur (Fe/S) clusters in all domains of life, and those of bacteria, animals, and protists have been shown to require a tetrahydrofolate to function. Two COG0354 proteins were identified in Arabidopsis and many other plants, one (At4g12130) related to those of α-proteobacteria and predicted to be mitochondrial, the other (At1g60990) related to those of cyanobacteria and predicted to be plastidial. Grasses and poplar appear to lack the latter. The predicted subcellular locations of the Arabidopsis proteins were validated by in vitro import assays with purified pea organelles and by targeting assays in Arabidopsis and tobacco protoplasts using green fluorescent protein fusions. The At4g12130 protein was shown to be expressed mainly in flowers, siliques, and seeds, whereas the At1g60990 protein was expressed mainly in young leaves. The folate dependence of both Arabidopsis proteins was established by functional complementation of an Escherichia coli COG0354 (ygfZ) deletant; both plant genes restored in vivo activity of the Fe/S enzyme MiaB but restoration was abrogated when folates were eliminated by deleting folP. Insertional inactivation of At4g12130 was embryo lethal; this phenotype was reversed by genetic complementation of the mutant. These data establish that COG0354 proteins have a folate-dependent function in mitochondria and plastids, and that the mitochondrial protein is essential. That plants retain mitochondrial and plastidial COG0354 proteins with distinct phylogenetic origins emphasizes how deeply the extant Fe/S cluster assembly machinery still reflects the ancient endosymbioses that gave rise to plants.

Evidence that glutamine transaminase and omega-amidase potentially act in tandem to close the methionine salvage cycle in bacteria and plants.

S-Adenosylmethionine is converted enzymatically and non-enzymatically to methylthioadenosine, which is recycled to methionine (Met) via a salvage pathway. In plants and bacteria, enzymes for all steps in this pathway are known except the last: transamination of α-ketomethylthiobutyrate to give Met. In mammals, glutamine transaminase K (GTK) and ω-amidase (ω-Am) are thought to act in tandem to execute this step, with GTK forming α-ketoglutaramate, which ω-Am hydrolyzes. Comparative genomics indicated that GTK and ω-Am could function likewise in plants and bacteria because genes encoding GTK and ω-Am homologs (i) co-express with the Met salvage gene 5-methylthioribose kinase in Arabidopsis, and (ii) cluster on the chromosome with each other and with Met salvage genes in diverse bacteria. Consistent with this possibility, tomato, maize, and Bacillus subtilis GTK and ω-Am homologs had the predicted activities: GTK was specific for glutamine as amino donor and strongly preferred α-ketomethylthiobutyrate as amino acceptor, and ω-Am strongly preferred α-ketoglutaramate. Also consistent with this possibility, plant GTK and ω-Am were localized to the cytosol, where the Met salvage pathway resides, as well as to organelles. This multiple targeting was shown to result from use of alternative start codons. In B. subtilis, ablating GTK or ω-Am had a modest but significant inhibitory effect on growth on 5-methylthioribose as sole sulfur source. Collectively, these data indicate that while GTK, coupled with ω-Am, is positioned to support significant Met salvage flux in plants and bacteria, it can probably be replaced by other aminotransferases.