Primary structure, partial purification and regulation of key enzymes of the acetyl cycle of arginine biosynthesis in Bacillus stearothermophilus: dual function of ornithine acetyltransferase. (original) (raw)
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Microbiology, 1996
A cluster of arginine biosynthetic genes of Corynebacterium glutamicum ATCC 13032, comprising argJ, argB and argD as well as part of argC and argF, has been cloned by heterologous complementation of an Escherichia coli argE mutant. The gene order has been established as argCJBDF by sequencing the entire 4.4 kb cloned DNA fragment. The C. glutamicum argB gene can be transcribed in E. coli cells from an internal promoter located in the coding part of the preceding argJ gene, whereas transcription of the argJ gene appears vector-dependent. Expression of the corynebacterial argB gene is repressed by arginine in the native host but not in recombinant E. coli cells. Feedback inhibition of the corresponding N-acetylglutamate kinase activity was observed both in cell extracts of C. glutamicum and in recombinant E. coli argB auxotrophic strains. Extracts of E. coli cells carrying cloned corynebacterial DNA display an ornithine acetyltransferase activity (encoded by argJ) which alleviates the acetylornithinase (encoded by argE) deficiency of the enterobacterial host. In contrast to Bacillus stearothermophilus ornithine acetyltransferase which also exhibits acetylglutamate synthase activity, C. glutamicum ornithine acetyltransferase appears monofunctional. ArgA and ArgB proteins from different sources share highly significant similarities. The evolutionary implications of these data are discussed.
European Journal of Biochemistry, 2000
The argJ gene coding for N2-acetyl-L-ornithine: L-glutamate N-acetyltransferase, the key enzyme involved in the acetyl cycle of L-arginine biosynthesis, has been cloned from thermophilic procaryotes: the archaeon Methanoccocus jannaschii, and the bacteria Thermotoga neapolitana and Bacillus stearothermophilus. Archaeal argJ only complements an Escherichia coli argE mutant (deficient in acetylornithinase, which catalyzes the fifth step in the linear biosynthetic pathway), whereas bacterial genes additionally complement an argA mutant (deficient in N-acetylglutamate synthetase, the first enzyme of the pathway). In keeping with these in vivo data the purified His-tagged ArgJ enzyme of M. jannaschii only catalyzes N2-acetylornithine conversion to ornithine, whereas T. neapolitana and B. stearothermophilus ArgJ also catalyze the conversion of glutamate to N-acetylglutamate using acetylCoA as the acetyl donor. M. jannaschii ArgJ is therefore a monofunctional enzyme, whereas T. neapolitana and B. stearothermophilus encoded ArgJ are bifunctional. Kinetic data demonstrate that in all three thermophilic organisms ArgJ-mediated catalysis follows ping-pong bi-bi kinetic mechanism. Acetylated ArgJ intermediates were detected in semireactions using [14C]acetylCoA or [14C]N2-acetyl-L-glutamate as acetyl donors. In this catalysis L-ornithine acts as an inhibitor; this amino acid therefore appears to be a key regulatory molecule in the acetyl cycle of L-arginine synthesis. Thermophilic ArgJ are synthesized as protein precursors undergoing internal cleavage to generate alpha and beta subunits which appear to assemble to alpha2beta2 heterotetramers in E. coli. The cleavage occurs between alanine and threonine residues within the highly conserved PXM-ATML motif detected in all available ArgJ sequences.
Applied and environmental microbiology, 1998
The goal of this work was to construct Escherichia coli strains capable of enhanced arginine production. The arginine biosynthetic capacity of previously engineered E. coli strains with a derepressed arginine regulon was limited by the availability of endogenous ornithine (M. Tuchman, B. S. Rajagopal, M. T. McCann, and M. H. Malamy, Appl. Environ. Microbiol. 63:33-38, 1997). Ornithine biosynthesis is limited due to feedback inhibition by arginine of N-acetylglutamate synthetase (NAGS), the product of the argA gene and the first enzyme in the pathway of arginine biosynthesis in E. coli. To circumvent this inhibition, the argA genes from E. coli mutants with feedback-resistant (fbr) NAGS were cloned into plasmids that contain "arg boxes," which titrate the ArgR repressor protein, with or without the E. coli carAB genes encoding carbamyl phosphate synthetase and the argI gene for ornithine transcarbamylase. The free arginine production rates of "arg-derepressed" E. ...
Journal of Bioscience and Bioengineering, 2009
Bacillus cereus strain 10-L-2 synthesizes two arylamine N-acetyltransferases (Nat-a and Nat-b) with broad substrate specificities toward aniline and its derivatives. In southern blot analysis using probes encoding the NH 2 -terminus of Nat-b and a conserved region of N-acetyltransferases, digested total DNA of strain 10-L-2 showed one positive band. We cloned and sequenced the gene encoding Nat-b. The NH 2 -terminal amino acid sequence predicted from the open reading frame (768 base pairs) corresponded to that of purified Nat-b. The cloned Nat-b gene was expressed in Escherichia coli. The expressed enzyme (BcNAT) from the recombinant strain was partially purified and characterized. Nat-b from strain 10-L-2 and BcNAT from the recombinant strain were slightly different from each others in substrate specificity and thermo-stability. We examined the biotransformations of 2-aminophenols and phenylenediamines by the whole cells of the recombinant strain. The cells converted these compounds into their corresponding acetanilides. Only one amino group of phenylenediamines was acetylated. The cells utilized 4-nitroacetanilide as an acetyl donor instead of acetyl-CoA. 4-Aminoacetanilide was produced and 4-nitroaniline was released almost stoichiometrically.
BMC Genomics, 2013
Background: Arginine biosynthesis in Corynebacterium glutamicum consists of eight enzymatic steps, starting with acetylation of glutamate, catalysed by N-acetylglutamate synthase (NAGS). There are different kinds of known NAGSs, for example, "classical" ArgA, bifunctional ArgJ, ArgO, and S-NAGS. However, since C. glutamicum possesses a monofunctional ArgJ, which catalyses only the fifth step of the arginine biosynthesis pathway, glutamate must be acetylated by an as of yet unknown NAGS gene. Results: Arginine biosynthesis was investigated by metabolome profiling using defined gene deletion mutants that were expected to accumulate corresponding intracellular metabolites. HPLC-ESI-qTOF analyses gave detailed insights into arginine metabolism by detecting six out of seven intermediates of arginine biosynthesis. Accumulation of N-acetylglutamate in all mutants was a further confirmation of the unknown NAGS activity. To elucidate the identity of this gene, a genomic library of C. glutamicum was created and used to complement an Escherichia coli ΔargA mutant. The plasmid identified, which allowed functional complementation, contained part of gene cg3035, which contains an acetyltransferase domain in its amino acid sequence. Deletion of cg3035 in the C. glutamicum genome led to a partial auxotrophy for arginine. Heterologous overexpression of the entire cg3035 gene verified its ability to complement the E. coli ΔargA mutant in vivo and homologous overexpression led to a significantly higher intracellular N-acetylglutamate pool. Enzyme assays confirmed the N-acetylglutamate synthase activity of Cg3035 in vitro. However, the amino acid sequence of Cg3035 revealed no similarities to members of known NAGS gene families.
Journal of General Microbiology, 1992
The expression of Buciffus steurothermophifus genes complementing arginine auxotrophs of Escherichiu coli was studied. The activity responsible for the formation of ornithine in B. steurothermophifus was identified as a repressible ornithine acetyltransferase (genetic symbol urgJ) encoded by the same DNA fragment as the argC, urgA and urgB genes. Buciffus subtilis and Buciffus ficheniformis displayed the same pattern of enzyme activities as B. steurothermophifus. In contrast to previous reports, these organisms consequently use the cyclic pathway of ornithine biosynthesis. B. steurothermophifus also pssesses a broad specificity aminoacylase which exhibits low affinity towards NZ-acetyl-L-ornithine.
Journal of Bacteriology, 2004
To help clarify the control of arginine synthesis in Thermotoga maritima, the putative gene (argB) for N-acetyl-L-glutamate kinase (NAGK) from this microorganism was cloned and overexpressed, and the resulting protein was purified and shown to be a highly thermostable and specific NAGK that is potently and selectively inhibited by arginine. Therefore, NAGK is in T. maritima the feedback control point of arginine synthesis, a process that in this organism involves acetyl group recycling and appears not to involve classical acetylglutamate synthase. The inhibition of NAGK by arginine was found to be pH independent and to depend sigmoidally on the concentration of arginine, with a Hill coefficient (N) of ϳ4, and the 50% inhibitory arginine concentration (I 0.5) was shown to increase with temperature, approaching above 65°C the I 0.50 observed at 37°C with the mesophilic NAGK of Pseudomonas aeruginosa (the best-studied arginine-inhibitable NAGK). At 75°C, the inhibition by arginine of T. maritima NAGK was due to a large increase in the K m for acetylglutamate triggered by the inhibitor, but at 37°C arginine also substantially decreased the V max of the enzyme. The NAGKs of T. maritima and P. aeruginosa behaved in gel filtration as hexamers, justifying the sigmoidicity and high Hill coefficient of arginine inhibition, and arginine or the substrates failed to disaggregate these enzymes. In contrast, Escherichia coli NAGK is not inhibited by arginine and is dimeric, and thus the hexameric architecture may be an important determinant of arginine sensitivity. Potential thermostability determinants of T. maritima NAGK are also discussed.
Effect of arginine on oligomerization and stability of N-acetylglutamate synthase
Scientific Reports, 2016
N-acetylglutamate synthase (NAGS; E.C.2.3.1.1) catalyzes the formation of N-acetylglutamate (NAG) from acetyl coenzyme A and glutamate. In microorganisms and plants, NAG is the first intermediate of the L-arginine biosynthesis; in animals, NAG is an allosteric activator of carbamylphosphate synthetase I and III. In some bacteria bifunctional N-acetylglutamate synthase-kinase (NAGS-K) catalyzes the first two steps of L-arginine biosynthesis. L-arginine inhibits NAGS in bacteria, fungi, and plants and activates NAGS in mammals. L-arginine increased thermal stability of the NAGS-K from Maricaulis maris (MmNAGS-K) while it destabilized the NAGS-K from Xanthomonas campestris (XcNAGS-K). Analytical gel chromatography and ultracentrifugation indicated tetrameric structure of the MmMNAGS-K in the presence and absence of L-arginine and a tetramer-octamer equilibrium that shifted towards tetramers upon binding of L-arginine for the XcNAGS-K. Analytical gel chromatography of mouse NAGS (mNAGS)...
Journal of Biological Chemistry, 2006
A Bacteroides fragilis gene (argF bf ), the disruption of which renders the bacterium auxotrophic for arginine, was expressed and its recombinant protein purified and studied. The novel protein catalyzes the carbamylation of N-succinyl-L-ornithine but not L-ornithine or N-acetyl-L-ornithine, forming N-succinyl-L-citrulline. Crystal structures of this novel transcarbamylase complexed with carbamyl phosphate and N-succinyl-L-norvaline, as well as sulfate and N-succinyl-L-norvaline have been determined and refined to 2.9 and 2.8 Å resolution, respectively. They provide structural evidence that this protein is a novel N-succinyl-L-ornithine transcarbamylase. The data provided herein suggest that B. fragilis uses N-succinyl-L-ornithine rather than N-acetyl-L-ornithine for de novo arginine biosynthesis and therefore that this pathway in Bacteroides is different from the canonical arginine biosynthetic pathway of most organisms. Comparison of the structures of the new protein with those recently reported for N-acetyl-L-ornithine transcarbamylase indicates that amino acid residue 90 (B. fragilis numbering) plays an important role in conferring substrate specificity for N-succinyl-L-ornithine versus N-acetyl-L-ornithine. Movement of the 120 loop upon substrate binding occurs in N-succinyl-L-ornithine transcarbamylase, while movement of the 80 loop and significant domain closure take place as in other transcarbamylases. These findings provide new information on the putative role of succinylated intermediates in arginine biosynthesis and on the evolution of transcarbamylases.