Characterization and kinetic mechanism of mono‐ and bifunctional ornithine acetyltransferases from thermophilic microorganisms

Abstract

The argJ gene coding for N 2 -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 N 2 -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 [ 14 C]acetylCoA or [ 14 C]N 2 -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 a and b subunits which appear to assemble to a2b2 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.

Surprising Arginine Biosynthesis: a Reappraisal of the Enzymology and Evolution of the Pathway in Microorganisms

“…When an OAT recycles the acetyl group, NAGK can become flow controlling; it is actually inhibited by arginine in many organisms, whether NAGS is present or not (11,12,59,71; also see below). For example, in Corynebacterium glutamicum and T. maritima, which have no recognizable NAGS or S-NAGS but an OAT (monofunctional in the former and probably bifunctional in the latter, by analogy with T. neapolitana [43]), arginine inhibits NAGK (see references 26 and 63, correcting an earlier report [72] for T. maritima). In P. aeruginosa, where both NAGS and OAT are present, both NAGS and NAGK are inhibited by arginine (34).…”

“…71,2007 SURPRISING ARGININE BIOSYNTHESIS 37 that some OATs could also synthesize acetylglutamate de novo from acetyl-CoA and glutamate (43,45). Not all OATs possess this dual activity, however: some are bifunctional whereas others do not use acetyl-CoA as the acetyl donor (43,76).…”

“…71,2007 SURPRISING ARGININE BIOSYNTHESIS 37 that some OATs could also synthesize acetylglutamate de novo from acetyl-CoA and glutamate (43,45). Not all OATs possess this dual activity, however: some are bifunctional whereas others do not use acetyl-CoA as the acetyl donor (43,76). In the most studied instance (Geobacillus [formerly Bacillus] stearothermophilus), the acetylation activity is about five times lower with acetyl-CoA as the substrate than with acetylornithine; moreover, in the former case, the reaction is irreversible, whereas the acetyl group exchange between acetylornithine and acetylglutamate is fully reversible (76).…”

“…Since the completion of a number of genome sequencing projects, a few instances indeed have emerged where no NAGS, but a bifunctional OAT, appears to be present. This is the case in B. subtilis (reinterpretation of old complementation data [55,81]), G. stearothermophilus, and Thermotoga spp., assuming that T. maritima, whose genome was completely sequenced, is similar to T. neapolitana, which has a bifunctional OAT (26,43). The lack of biochemical information on the catalytic properties of most genomically identified OATs precludes further identification.…”

Genetic and biochemical characterization of arginine biosynthesis in Sinorhizobium meliloti 1021

“…Other ArgJs have been shown to undergo autoproteolysis, resulting in a and b polypeptides containing the N and C terminal portions, respectively, of the enzyme (Kershaw et al, 2002;Marc et al, 2000). S. meliloti ArgJ purified as two peptides with molecular masses of 23 and 25 kDa (Table 3), consistent with autoproteolysis occurring between A194/T195, which would generate a and b polypeptides with predicted masses of 20 and 23 kDa, respectively.…”