A novel type of N-acetylglutamate synthase is involved in the first step of arginine biosynthesis in Corynebacterium glutamicum

Abstract

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.

Conclusions: The N-acetylglutamate synthase Cg3035 is able to catalyse the first step of arginine biosynthesis in C. glutamicum. It represents a novel class of NAGS genes apparently present only in bacteria of the suborder Corynebacterineae, comprising amongst others the genera Corynebacterium, Mycobacterium, and Nocardia. Therefore, the name C-NAGS (Corynebacterineae-type NAGS) is proposed for this new family.

Figure 1

General pathway of arginine biosynthesis in prokaryotes including two known routes for removal of the acetyl group. Vertical arrows represent the linear pathway, whereas the alternative cyclic pathway in which the acetyl group is recycled by ornithine acetyltransferase (encoded by argJ) is indicated by a dashed arrow. Intermediates and immediate precursors are given in bold letters, enzymes are in boxes. ArgJ* designates bifunctional proteins. Abbreviations: HS-CoA = coenzyme A; Ac-CoA = acetyl-CoA; (P)P_i = (pyro)phosphate; HCO₃- = bicarbonate.

Figure 2

Bar charts of normalised peak areas of six intermediates of arginine biosynthesis after HPLC-ESI-qTOF analysis.C. glutamicum ATCC 13032 (WT) and seven double deletion mutants were cultivated with l-arginine until exponential phase. Then l-arginine was removed and cells were further incubated to accumulate intracellular metabolites. The boxes in each diagram indicate the respective intracellular compound and its mass-to-charge ratio. Peak detection and integration was performed on base peak chromatograms (BPC) of m/z-values of [M + H]⁺ ions. Values that are significantly different from the wildtype level (Student’s T-test p < 0.05) are indicated by an asterisk. Error bars represent standard deviations of four biological replicates.

Figure 3

Genomic map of the chromosomal region of C. glutamicum carrying cg3035.Cg3035 is indicated as dark grey arrow, adjacent ORFs as light grey arrows. The cloned region of the complementation plasmid is shown as dark grey box. Also depicted are the binding positions of primers (small open arrows) used to generate the deletion construct (light grey boxes). The deleted region is depicted as empty box.

Figure 4

Diagram of normalised peak areas of N-acetylglutamate in different C. glutamicum strains. Hydrophilic metabolites were extracted from C. glutamicum ATCC 13032 (WT), ∆cg3035 as well as WT pZ8-1 (empty vector) and WT pZ8-1::cg3035. Peak detection and integration was performed on base peak chromatograms of m/z-values of [M + H]⁺ ions. Error bars represent standard deviations of four biological replicates. Values that are significantly different from the wildtype level (Student’s T-test p < 0.01) are indicated by an asterisk.

Figure 5

Phylogenetic analysis of Cg3035 relatives in other organisms and genomic context of cg3035 in different bacteria. A) Cg3035 was used as query against the RefSeq database of BLASTP to identify similar proteins. The most likely orthologues of Cg3035 were aligned using COBALT [35] and a Fast-Minimum-Evolution tree was built with this software. B) The freeware tool GeConT II (http://bioinfo.ibt.unam.mx/gecont/index.cgi) was used to visualise the genomic context of cg3035 and its orthologous genes within fully sequenced bacterial genomes [37]. Species names abbreviated as in http://www.expasy.ch/cgi-bin/speclist.