Bioinformatic analysis of an unusual gene-enzyme relationship in the arginine biosynthetic pathway among marine gamma proteobacteria: implications concerning the formation of N-acetylated intermediates in prokaryotes

Abstract

Background: The N-acetylation of L-glutamate is regarded as a universal metabolic strategy to commit glutamate towards arginine biosynthesis. Until recently, this reaction was thought to be catalyzed by either of two enzymes: (i) the classical N-acetylglutamate synthase (NAGS, gene argA) first characterized in Escherichia coli and Pseudomonas aeruginosa several decades ago and also present in vertebrates, or (ii) the bifunctional version of ornithine acetyltransferase (OAT, gene argJ) present in Bacteria, Archaea and many Eukaryotes. This paper focuses on a new and surprising aspect of glutamate acetylation. We recently showed that in Moritella abyssi and M. profunda, two marine gamma proteobacteria, the gene for the last enzyme in arginine biosynthesis (argH) is fused to a short sequence that corresponds to the C-terminal, N-acetyltransferase-encoding domain of NAGS and is able to complement an argA mutant of E. coli. Very recently, other authors identified in Mycobacterium tuberculosis an independent gene corresponding to this short C-terminal domain and coding for a new type of NAGS. We have investigated the two prokaryotic Domains for patterns of gene-enzyme relationships in the first committed step of arginine biosynthesis.

Results: The argH-A fusion, designated argH(A), and discovered in Moritella was found to be present in (and confined to) marine gamma proteobacteria of the Alteromonas- and Vibrio-like group. Most of them have a classical NAGS with the exception of Idiomarina loihiensis and Pseudoalteromonas haloplanktis which nevertheless can grow in the absence of arginine and therefore appear to rely on the arg(A) sequence for arginine biosynthesis. Screening prokaryotic genomes for virtual argH-X 'fusions' where X stands for a homologue of arg(A), we retrieved a large number of Bacteria and several Archaea, all of them devoid of a classical NAGS. In the case of Thermus thermophilus and Deinococcus radiodurans, the arg(A)-like sequence clusters with argH in an operon-like fashion. In this group of sequences, we find the short novel NAGS of the type identified in M. tuberculosis. Among these organisms, at least Thermus, Mycobacterium and Streptomyces species appear to rely on this short NAGS version for arginine biosynthesis.

Conclusion: The gene-enzyme relationship for the first committed step of arginine biosynthesis should now be considered in a new perspective. In addition to bifunctional OAT, nature appears to implement at least three alternatives for the acetylation of glutamate. It is possible to propose evolutionary relationships between them starting from the same ancestral N-acetyltransferase domain. In M. tuberculosis and many other bacteria, this domain evolved as an independent enzyme, whereas it fused either with a carbamate kinase fold to give the classical NAGS (as in E. coli) or with argH as in marine gamma proteobacteria. Moreover, there is an urgent need to clarify the current nomenclature since the same gene name argA has been used to designate structurally different entities. Clarifying the confusion would help to prevent erroneous genomic annotation.

Figure 1

The de novo arginine biosynthetic pathway. Outline of the pathway; for each step (labeled by a number in a square) the following features are indicated: gene name, EC number, enzyme name and its abbreviation.

Figure 2

Reactions involving L-glutamate. This figure has been drawn using the tools available at Metacyc [37].

Figure 3

Phylogenetic relationships of species harbouring ArgH(A) fusions. A simplified version of the 16S rRNA phylogenetic tree of Alteromonas- and Vibrio-like bacteria reconstructed by Ivanova et al.[11] has been drawn and enriched with the following information. On the right-hand side, available genomic information concerning arg genes; ppc refers to the gene for phosphoenolpyruvate carboxylase, adjacent to argE in many of these organisms; nd: not determined. On the left-hand side, the putative content of the ancestral arg gene clusters are indicated for each deep node of this tree.

Figure 4

Phylogenetic tree of the arg(A) homologues. The sequences have been collected, multiply aligned and used to reconstruct an evolutionary tree as described in Methods. The tree has been rooted using the acetyltransferase domain of NAGS sequences as an outgroup (subtree indicated with yellow color). The two other subtrees corresponding to the Arg(A) and ArgX sequences are indicated with pink and blue colors, respectively. Species names are according to SwissProt conventions (the detailed list is shown in Table 1).

Figure 5

Phylogenetic tree of the ArgH(A) and ArgHX fusions. The sequences have been collected, multiply aligned and used to reconstruct an evolutionary tree as described in Methods. The grey circles indicate the bootstrap values for the deep nodes which are less than 60%. Species names are according to SwissProt conventions (the detailed list is shown in Table 1).

Figure 6

Conserved motifs in putative glutamate N-acetyltransferases. The homologues to the (A) domain of ArgH(A) of M. abyssi and belonging to six ArgH(A), three ArgHX and two ArgA proteins have been multiply aligned and edited in BioEdit software [32]. A consensus sequence has been computed. Very strongly conserved residues(letters) and moderately conserved ones (stars) are indicated. The four motifs containing the residues identified by comparative analysis of N-acetyltransferases which use acetylCoA as donor of the acetyl group [20] and references therein] are underlined by thick lines and numbered. Species names are according to SwissProt conventions.

Figure 7

Evolution of glutamate acetylation from a primordial N- acetyltransferase. The recruitment of a primordial N-acetyltransferase for L-glutamate acetylation in the first step of arginine biosynthesis has been made according to at least three different evolutionary ways. The events of gene duplication and gene fusion that allowed evolution toward either the two-domain N-acetylglutamate synthase (EC 2.3.1.1) or the bifunctional argH(A) fusion are schematized. This scheme does not specify whether the argH(A) fusion arose in an organism originally devoid of NAGS or in an organism having lost NAGS. The yellow domains in contemporary proteins (bottom line) are bearing the ArgA activity (EC 2.3.1.1). The contemporary proteins that have been experimentally studied are indicated between brackets.