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Aspartate aminotransferase Rv3722c governs aspartate-dependent nitrogen metabolism in Mycobacterium tuberculosis.

ABSTRACT: Gene rv3722c of Mycobacterium tuberculosis is essential for in vitro growth, and encodes a putative pyridoxal phosphate-binding protein of unknown function. Here we use metabolomic, genetic and structural approaches to show that Rv3722c is the primary aspartate aminotransferase of M. tuberculosis, and mediates an essential but underrecognized role in metabolism: nitrogen distribution. Rv3722c deficiency leads to virulence attenuation in macrophages and mice. Our results identify aspartate biosynthesis and nitrogen distribution as potential species-selective drug targets in M. tuberculosis.

SUBMITTER: Jansen RS

PROVIDER: S-EPMC7181641 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Aspartate aminotransferase Rv3722c governs aspartate-dependent nitrogen metabolism in Mycobacterium tuberculosis.

Jansen Robert S RS Mandyoli Lungelo L Hughes Ryan R Wakabayashi Shoko S Pinkham Jessica T JT Selbach Bruna B Guinn Kristine M KM Rubin Eric J EJ Sacchettini James C JC Rhee Kyu Y KY

Nature communications 20200423 1

Gene rv3722c of Mycobacterium tuberculosis is essential for in vitro growth, and encodes a putative pyridoxal phosphate-binding protein of unknown function. Here we use metabolomic, genetic and structural approaches to show that Rv3722c is the primary aspartate aminotransferase of M. tuberculosis, and mediates an essential but underrecognized role in metabolism: nitrogen distribution. Rv3722c deficiency leads to virulence attenuation in macrophages and mice. Our results identify aspartate biosyn ...[more]

PMID: 32327655

Similar Datasets

Aspartate aminotransferase Rv3722c governs aspartate-dependent nitrogen metabolism in Mycobacterium tuberculosis

Project description:<p>Gene rv3722c of Mycobacterium tuberculosis is essential for in vitro growth, and encodes a putative pyridoxal phosphate-binding protein of unknown function. Here we use metabolomic, genetic and structural approaches to show that Rv3722c is the primary aspartate aminotransferase of M. tuberculosis, and mediates an essential but underrecognized role in metabolism: nitrogen distribution. Rv3722c deficiency leads to virulence attenuation in macrophages and mice. Our results identify aspartate biosynthesis and nitrogen distribution as potential species-selective drug targets in M. tuberculosis.</p>

2020-05-01 | MTBLS1559 | MetaboLights

Mycobacterium tuberculosis nitrogen assimilation and host colonization require aspartate.

Project description:Here we identify the amino acid transporter AnsP1 as the unique aspartate importer in the human pathogen Mycobacterium tuberculosis. Metabolomic analysis of a mutant with an inactive AnsP1 revealed that the transporter is essential for M. tuberculosis to assimilate nitrogen from aspartate. Virulence of the AnsP1 mutant is impaired in vivo, revealing that aspartate is a primary nitrogen source required for host colonization by the tuberculosis bacillus.

| S-EPMC3856356 | biostudies-literature

Overexpression, purification, crystallization and structure determination of AspB, a putative aspartate aminotransferase from Mycobacterium tuberculosis.

Project description:A recombinant version of a putative aspartate aminotransferase, AspB (encoded by the ORF Rv3565), from Mycobacterium tuberculosis (Mtb) was overexpressed in M. smegmatis and purified to homogeneity using liquid chromatography. Crystals of AspB were grown in a condition consisting of 0.2?M ammonium phosphate monobasic, 0.1?M calcium chloride dihydrate employing the hanging-drop vapour-diffusion method at 298?K. The crystals diffracted to a limit of 2.50?Å resolution and belonged to the orthorhombic space group P2?2?2?, with unit-cell parameters a=93.27, b=98.19, c=198.70?Å. The structure of AspB was solved by the molecular-replacement method using a putative aminotransferase from Silicibacter pomeroyi (PDB entry 3h14) as the search model. The template shares 46% amino-acid sequence identity with Mtb AspB. The crystal asymmetric unit contains four AspB molecules (the Mr of each is 42,035?Da).

| S-EPMC4089534 | biostudies-literature

Light-enhanced catalysis by pyridoxal phosphate-dependent aspartate aminotransferase.

Project description:The mechanisms of pyridoxal 5'-phosphate (PLP)-dependent enzymes require substrates to form covalent "external aldimine" intermediates, which absorb light strongly between 410 and 430 nm. Aspartate aminotransferase (AAT) is a prototypical PLP-dependent enzyme that catalyzes the reversible interconversion of aspartate and ?-ketoglutarate with oxalacetate and glutamate. From kinetic isotope effects studies, it is known that deprotonation of the aspartate external aldimine C(?)-H bond to give a carbanionic quinonoid intermediate is partially rate limiting in the thermal AAT reaction. We show that excitation of the 430-nm external aldimine absorption band increases the steady-state catalytic activity of AAT, which is attributed to the photoenhancement of C(?)-H deprotonation on the basis of studies with Schiff bases in solution. Blue light (250 mW) illumination gives an observed 2.3-fold rate enhancement for WT AAT activity, a 530-fold enhancement for the inactive K258A mutant, and a 58600-fold enhancement for the PLP-Asp Schiff base in water. These different levels of enhancement correlate with the intrinsic reactivities of the C(?)-H bond in the different environments, with the less reactive Schiff bases exhibiting greater enhancement. Time-resolved spectroscopy, ranging from femtoseconds to minutes, was used to investigate the nature of the photoactivation of C(?)-H bond cleavage in PLP-amino acid Schiff bases both in water and bound to AAT. Unlike the thermal pathway, the photoactivation pathway involves a triplet state with a C(?)-H pK(a) that is estimated to be between 11 and 19 units lower than the ground state for the PLP-Val Schiff base in water.

| S-EPMC3021986 | biostudies-literature

The role of glutamine oxoglutarate aminotransferase and glutamate dehydrogenase in nitrogen metabolism in Mycobacterium bovis BCG.

Project description:Recent evidence suggests that the regulation of intracellular glutamate levels could play an important role in the ability of pathogenic slow-growing mycobacteria to grow in vivo. However, little is known about the in vitro requirement for the enzymes which catalyse glutamate production and degradation in the slow-growing mycobacteria, namely; glutamine oxoglutarate aminotransferase (GOGAT) and glutamate dehydrogenase (GDH), respectively. We report that allelic replacement of the Mycobacterium bovis BCG gltBD-operon encoding for the large (gltB) and small (gltD) subunits of GOGAT with a hygromycin resistance cassette resulted in glutamate auxotrophy and that deletion of the GDH encoding-gene (gdh) led to a marked growth deficiency in the presence of L-glutamate as a sole nitrogen source as well as reduction in growth when cultured in an excess of L-asparagine.

| S-EPMC3868603 | biostudies-literature

Chemical Mechanism of the Branched-Chain Aminotransferase IlvE from Mycobacterium tuberculosis.

Project description:The biosynthetic pathway of the branched-chain amino acids is essential for Mycobacterium tuberculosis growth and survival. We report here the kinetic and chemical mechanism of the pyridoxal 5'-phosphate (PLP)-dependent branched-chain aminotransferase, IlvE, from M. tuberculosis (MtIlvE). This enzyme is responsible for the final step of the synthesis of the branched-chain amino acids isoleucine, leucine, and valine. As seen in other aminotransferases, MtIlvE displays a ping-pong kinetic mechanism. pK values were identified from the pH dependence on V as well as V/K, indicating that the phosphate ester of the PLP cofactor, and the ?-amino group from l-glutamate and the active site Lys204, play roles in acid-base catalysis and binding, respectively. An intrinsic primary kinetic isotope effect was identified for the ?-C-H bond cleavage of l-glutamate. Large solvent kinetic isotope effect values for the ping and pong half-reactions were also identified. The absence of a quininoid intermediate in combination with the Dkobs in our multiple kinetic isotope effects under single-turnover conditions suggests a concerted type of mechanism. The deprotonation of C2 of l-glutamate and the protonation of C4' of the PLP cofactor happen synchronously in the ping half-reaction. A chemical mechanism is proposed on the basis of the results obtained here.

| S-EPMC5443349 | biostudies-literature

Chemoinformatic identification of novel inhibitors against Mycobacterium tuberculosis L-aspartate ?-decarboxylase.

Project description:L-aspartate ?-decarboxylase (ADC) belongs to a class of pyruvoyl dependent enzymes and catalyzes the conversion of aspartate to ?-alanine in the pantothenate pathway, which is critical for the growth of several micro-organisms, including Mycobacterium tuberculosis (Mtb). Its presence only in micro-organisms, fungi and plants and its absence in animals, particularly human, make it a promising drug target. We have followed a chemoinformatics-based approach to identify potential drug-like inhibitors against Mycobacterium tuberculosis L-aspartate ?-decarboxylase (MtbADC). The structure-based high throughput virtual screening (HTVS) mode of the Glide program was used to screen 333,761 molecules of the Maybridge, National Cancer Institute (NCI) and Food and Drug Administration (FDA) approved drugs databases. Ligands were rejected if they cross-reacted with S-adenosylmethionine (SAM) decarboxylase, a human pyruvoyl dependent enzyme. The lead molecules were further analyzed for physicochemical and pharmacokinetic parameters, based on Lipinski's rule of five, and ADMET (absorption, distribution, metabolism, excretion and toxicity) properties. This analysis resulted in eight small potential drug-like inhibitors that are in agreement with the binding poses of the crystallographic ADC:fumarate and ADC:isoasparagine complex structures and whose backbone scaffolds seem to be suitable for further experimental studies in therapeutic development against tuberculosis.

| S-EPMC3314653 | biostudies-literature

Derailing the aspartate pathway of Mycobacterium tuberculosis to eradicate persistent infection.

Project description:A major constraint for developing new anti-tuberculosis drugs is the limited number of validated targets that allow eradication of persistent infections. Here, we uncover a vulnerable component of Mycobacterium tuberculosis (Mtb) persistence metabolism, the aspartate pathway. Rapid death of threonine and homoserine auxotrophs points to a distinct susceptibility of Mtb to inhibition of this pathway. Combinatorial metabolomic and transcriptomic analysis reveals that inability to produce threonine leads to deregulation of aspartate kinase, causing flux imbalance and lysine and DAP accumulation. Mtb's adaptive response to this metabolic stress involves a relief valve-like mechanism combining lysine export and catabolism via aminoadipate. We present evidence that inhibition of the aspartate pathway at different branch-point enzymes leads to clearance of chronic infections. Together these findings demonstrate that the aspartate pathway in Mtb relies on a combination of metabolic control mechanisms, is required for persistence, and represents a target space for anti-tuberculosis drug development.

| S-EPMC6746716 | biostudies-literature

Cloning and nucleotide sequencing of Rhizobium meliloti aminotransferase genes: an aspartate aminotransferase required for symbiotic nitrogen fixation is atypical.

Project description:In Rhizobium meliloti, an aspartate aminotransferase (AspAT) encoded within a 7.3-kb HindIII fragment was previously shown to be required for symbiotic nitrogen fixation and aspartate catabolism (V. K. Rastogi and R.J. Watson, J. Bacteriol. 173:2879-2887, 1991). A gene coding for an aromatic aminotransferase located within an 11-kb HindIII fragment was found to complement the AspAT deficiency when overexpressed. The genes encoding these two aminotransferases, designated aatA and tatA, respectively, have been localized by subcloning and transposon Tn5 mutagenesis. Sequencing of the tatA gene revealed that it encodes a protein homologous to an Escherichia coli aromatic aminotransferase and most of the known AspAT enzymes. However, sequencing of the aatA gene region revealed two overlapping open reading frames, neither of which encoded an enzyme with homology to the typical AspATs. Polymerase chain reaction was used to selectively generate one of the candidate sequences for subcloning. The cloned fragment complemented the original nitrogen fixation and aspartate catabolism defects and was shown to encode an AspAT with the expected properties. Sequence analysis showed that the aatA protein has homology to AspATs from two thermophilic bacteria and the eukaryotic tyrosine aminotransferases. These aminotransferases form a distinct class in which only 13 amino acids are conserved in comparison with the well-known AspAT family. DNA homologous to the aatA gene was found to be present in Agrobacterium tumefaciens and other rhizobia but not in Klebsiella pneumoniae or E. coli.

| S-EPMC204262 | biostudies-other

Nitrogen-efficient rice over-expressing alanine aminotransferase

Project description:Oryza sativa cv. Nipponbare was engineered to over-express a barley alanine aminotransferase (alaAT) gene using the promoter (OsANT1) from a rice aldehyde dehydrogenase gene that expresses in roots. We are using biotechnology to improve the nitrogen use efficiency of rice by over-expressing alaAT in a tissue specific (root) manner. The AlaAT enzyme is a reversible aminotransferase that is linked to both C and N metabolism since it uses pyruvate plus glutamate to produce alanine and 2-oxoglutarate, and visa versa.

2012-07-27 | GSE39687 | GEO

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