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Abstract The existing treatment regime against tuberculosis is not adequate, and novel therapeutic interventions are required to target Mycobacterium tuberculosis (Mtb) pathogenesis. We report Pranlukast (PRK) as a novel allosteric inhibitor of Mtb's arginine biosynthetic enzyme, Ornithine acetyltransferase (MtArgJ). PRK treatment remarkably abates the survival of free as well as macrophage‐internalized Mtb, and shows enhanced efficacy in combination with standard‐of‐care drugs. Notably, PRK also reduces the 5‐lipoxygenase (5‐LO) signaling in the infected macrophages, thereby surmounting an enhanced response against intracellular pathogen. Further, treatment with PRK alone or with rifampicin leads to significant decrease in Mtb burden and tubercular granulomas in Mtb‐infected mice lungs. Taken together, this study demonstrates a novel allosteric inhibitor of MtArgJ, which acts as a dual‐edged sword, by targeting the intracellular bacteria as well as the bacterial pro‐survival signaling in the host. PRK is highly effective against in vitro and in vivo survival of Mtb and being an FDA‐approved drug, it shows a potential for development of advanced combinatorial therapy against tuberculosis.

L-Ornithine is a non-protein amino acid with extensive applications in the food and pharmaceutical industries. In this study, we performed metabolic pathway engineering of an L-arginine hyper-producing strain of Corynebacterium crenatum for L-ornithine production. First, we amplified the L-ornithine biosynthetic pathway flux by blocking the competing branch of the pathway. To enhance L-ornithine synthesis, we performed site-directed mutagenesis of the ornithine-binding sites to solve the problem of L-ornithine feedback inhibition for ornithine acetyltransferase. Alternatively, the genes argA from Escherichia coli and argE from Serratia marcescens, encoding the enzymes N-acetyl glutamate synthase and N-acetyl-L-ornithine deacetylase, respectively, were introduced into Corynebacterium crenatum to mimic the linear pathway of L-ornithine biosynthesis. Fermentation of the resulting strain in a 5-L bioreactor allowed a dramatically increased production of L-ornithine, 40.4 g/L, with an overall productivity of 0.673 g/L/h over 60 h. This demonstrates that an increased level of transacetylation is beneficial for L-ornithine biosynthesis. [ABSTRACT FROM AUTHOR]

Abstract: The existing treatment regime against tuberculosis is not adequate, and novel therapeutic interventions are required to target Mycobacterium tuberculosis (Mtb) pathogenesis. We report Pranlukast (PRK) as a novel allosteric inhibitor of Mtb's arginine biosynthetic enzyme, Ornithine acetyltransferase (MtArgJ). PRK treatment remarkably abates the survival of free as well as macrophage‐internalized Mtb, and shows enhanced efficacy in combination with standard‐of‐care drugs. Notably, PRK also reduces the 5‐lipoxygenase (5‐LO) signaling in the infected macrophages, thereby surmounting an enhanced response against intracellular pathogen. Further, treatment with PRK alone or with rifampicin leads to significant decrease in Mtb burden and tubercular granulomas in Mtb‐infected mice lungs. Taken together, this study demonstrates a novel allosteric inhibitor of MtArgJ, which acts as a dual‐edged sword, by targeting the intracellular bacteria as well as the bacterial pro‐survival signaling in the host. PRK is highly effective against in vitro and in vivo survival of Mtb and being an FDA‐approved drug, it shows a potential for development of advanced combinatorial therapy against tuberculosis. [ABSTRACT FROM AUTHOR]

l-Ornithine is an intermediate of the l-arginine biosynthetic pathway in Corynebacterium glutamicum. The effect of ornithine acetyltransferase (OATase; ArgJ) on l-ornithine production was investigated, and C. glutamicum 1006 was engineered to overproduce l-ornithine as a major product by inactivating regulatory repressor argR gene and overexpressing argJ gene. A genome sequence analysis indicated that the argF gene encoding ornithine carbamoyltransferase in C. glutamicum 1006 was mutated, resulting in the accumulation of a certain amount of l-ornithine (20.5 g/L). The assays using a crude extract of C. glutamicum 1006 indicated that the l-ornithine concentration for 50% inhibition of OAT was 5 mM. To enhance l-ornithine production, the argJ gene from C. glutamicum ATCC 13032 was overexpressed. In flask cultures, the resulting strain, C. glutamicum 1006∆ argR-argJ, produced 31.6 g/L l-ornithine, which is 54.15% more than that produced by C. glutamicum 1006. The OAT activity of C. glutamicum 1006∆ argR- argJ was significantly greater than that of C. glutamicum 1006, and this study achieved the highest conversion ratio of sugar to acid (0.396 g/g) compared with those of previous reports. ArgJ strongly influences the production of l-ornithine in C. glutamicum. [ABSTRACT FROM AUTHOR]

In this study, Corynebacterium glutamicum ATCC 13032 was engineered to produce l-citrulline through a metabolic engineering strategy. To prevent the flux away from l-citrulline and to increase the expression levels of genes involved in the citrulline biosynthesis pathway, the argininosuccinate synthase gene ( argG) and the repressor gene ( argR) were inactivated. The engineered C. glutamicum ATCC 13032 ∆ argG ∆ argR (CIT 2) produced higher amounts of l-citrulline (5.43 g/L) compared to the wildtype strain (0.15 g/L). To determine new strategies for further enhancement of l-citrulline production, the effect of l-citrulline on ornithine acetyltransferase (EC 2.3.1.35; OATase; ArgJ) was first investigated. Citrulline was determined to inhibit Ornithine acetyltransferase; for 50 % inhibition, citrulline concentration was 30 mM. The argJ gene from C. glutamicum ATCC 13032 was cloned, and the recombinant shuttle plasmid pXMJ19- argJ was constructed and expressed in C. glutamicum ATCC 13032 ∆ argG ∆ argR (CIT 2). Overexpression of the argJ gene exhibited increased OAT activity and resulted in a positive effect on citrulline production (8.51 g/L). These results indicate that OAT plays a vital role during l-citrulline production in C. glutamicum. [ABSTRACT FROM AUTHOR]

Abstract: Mycobacterium tuberculosis ornithine acetyltransferase (Mtb OAT; E.C. 2.3.1.35) is a key enzyme of the acetyl recycling pathway during arginine biosynthesis. It reversibly catalyzes the transfer of the acetyl group from N-acetylornithine (NAORN) to l-glutamate. Mtb OAT is a member of the N-terminal nucleophile fold family of enzymes. The crystal structures of Mtb OAT in native form and in its complex with ornithine (ORN) have been determined at 1.7 and 2.4 Å resolutions, respectively. ORN is a competitive inhibitor of this enzyme against l-glutamate as substrate. Although the acyl–enzyme complex of Streptomyces clavuligerus ornithine acetyltransferase has been determined, ours is the first crystal structure to be reported of an ornithine acetyltransferase in complex with an inhibitor. ORN binding does not alter the structure of Mtb OAT globally. However, its presence stabilizes the three C-terminal residues that are disordered and not observed in the native structure. Also, stabilization of the C-terminal residues by ORN reduces the size of the active-site pocket volume in the structure of the ORN complex. The interactions of ORN and the protein residues of Mtb OAT unambiguously delineate the active-site residues of this enzyme in Mtb. Moreover, modeling studies carried out with NAORN based on the structure of the ORN–Mtb OAT complex reveal important interactions of the carbonyl oxygen of the acetyl group of NAORN with the main-chain nitrogen atom of Gly128 and with the side-chain oxygen of Thr127. These interactions likely help in the stabilization of oxyanion formation during enzymatic reaction and also will polarize the carbonyl carbon–oxygen bond, thereby enabling the side-chain atom Oγ1 of Thr200 to launch a nucleophilic attack on the carbonyl-carbon atom of the acetyl group of NAORN. [Copyright &y& Elsevier]

The gene product of open reading frame Rv1653 from Mycobacterium tuberculosis is annotated as encoding a probable ornithine acetyltransferase (OATase; EC 2.3.1.35), an enzyme that catalyzes two steps in the arginine-biosynthesis pathway. It transfers an acetyl group from N-acetylornithine to l-glutamate to produce N-acetylglutamate and l-ornithine. Rv1653 was crystallized using the sitting-drop vapour-diffusion method. The native crystals diffracted to a resolution of 1.7 Å and belonged to space group P212121, with unit-cell parameters a = 60.1, b = 99.7, c = 155.3 Å. The preliminary X-ray study showed the presence of a dimer in the asymmetric unit of the crystals, which had a Matthews coefficient VM of 2.8 Å3 Da−1. [ABSTRACT FROM AUTHOR]

A study is presented on the crystallization of ornithine acetyltransferase from yeast, which catalyzes the fifth step in microbial arginine synthesis. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either the batch or hanging-drop techniques. This makes the difference between useless crystals and crystals that allow successful determination of the structure of the protein. The crystals belong to space group P4, with unit-cell parameters a = b = 66.98, c = 427.09 Å, and a data set was collected to 2.76 Å. [ABSTRACT FROM AUTHOR]

The clinically used beta-lactamase inhibitor clavulanic acid is produced by fermentation of Streptomyces clavuligerus . The orf6 gene of the clavulanic acid biosynthetic gene cluster in S. clavuligerus encodes a protein that shows sequence homology to ornithine acetyltransferase (OAT), the fifth enzyme of the arginine biosynthetic pathway. Orf6 was overexpressed in Escherichia coli (at ≈ 15% of total soluble protein by SDS/PAGE analysis) indicating it was not toxic to the host cells. The recombinant protein was purified (to > 95% purity) by a one-step technique. Like other OATs it was synthesized as a precursor protein which underwent autocatalytic internal cleavage in E. coli to generate α and β subunits. Cleavage was shown to occur between the alanine and threonine residues in a KGXGMXXPX--(M/L)AT(M/L)L motif conserved within all identified OAT sequences. Gel filtration and native electrophoresis analyses implied that the ORF6 protein was an α2 β2 heterotetramer and direct evidence for this came from mass spectrometric analyses. Although anomalous migration of the β subunit was observed by standard SDS/PAGE analysis, which indicated the presence of two bands (as previously observed for other OATs), mass spectrometric analyses did not reveal any evidence for post-translational modification of the β subunit. Extended denaturation with SDS before PAGE resulted in observation of a single major β subunit band. Purified ORF6 was able to catalyse the reversible transfer of an acetyl group from N -acetylornithine to glutamate, but not the formation of N -acetylglutamate from glutamate and acetyl-coenzyme A, nor (detectably) the hydrolysis of N -acetylornithine. Mass spectrometry also revealed the reaction proceeds via acetylation of the β subunit. [ABSTRACT FROM AUTHOR]

We have cloned by functional complementation and characterized the yeast ARG7 gene encoding mitochondrial ornithine acetyltransferase, the enzyme catalyzing the fifth step in arginine biosynthesis. While forming ornithine, this enzyme regenerates acetylglutamate, also produced in the first step by the ARG2-encoded acetylglutamate synthase. Interestingly, total deletion of the genomic ARG7 ORF resulted in an arginine-leaky phenotype, indicating that yeast cells possess an alternative route for generating ornithine from acetylornithine. Yeast ornithine acetyltransferase has been purified and characterized previously as a heterodimer of two subunits proposed to derive from a single precursor protein [Liu, Y.-S., Van Heeswijck R., Hoj. P. & Hoogenraad, N. (1995) Eur J. Biochem. 228, 291–296]; those authors further suggested that the internal processing of Arg7p, which is a mitochondrial enzyme, might occur in the matrix, while the leader peptide would be of the non-cleavable type. The characterization of the gene (a) establishes that Arg7p is indeed encoded by a single gene, (b) demonstrates the existence of a cleaved mitochondrial prepeptide of eight residues, and (c) shows that the predicted internal processing site is unlike the mitochondrial proteolytic peptidase target sequence. Yeast Arg7p shares between 32–43% identity in pairwise comparisons with the ten analogous bacterial ArgJ enzymes characterized. Among these evolutionarily related enzymes, some but not all appear bifunctional, being able to produce acetylglutamate not only from acetylornithine but also from acetyl- CoA, thus catalyzing the same reaction as the apparently unrelated acetylglutamate synthase. We have addressed the question of the bifunctionality of the eucaryotic enzyme, showing that overexpressed ARG7 can complement yeast arg2 and Escherichia coli argA mutations (affecting acetylglutamate synthase). Furthermore, Arg7p-linked acetylglutamate synthase activity was measurable in an assay. The yeast enzyme is thus clearly, albeit modestly, bifunctional. As with several bacterial ornithine acetyltransferases, the activity of Arg7p was practically insensitive to arginine but strongly inhibited by ornithine, which behaved as a competitive inhibitor. [ABSTRACT FROM AUTHOR]