Petri Kathrin, Walter Frederik, Persicke Marcus, Rückert Christian, Kalinowski Jörn
Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27 33615 Bielefeld, Germany.
BMC Genomics. 2013 Oct 18;14:713. doi: 10.1186/1471-2164-14-713.
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.
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.
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.
谷氨酸棒杆菌中的精氨酸生物合成由八个酶促步骤组成，起始于谷氨酸的乙酰化反应，该反应由N - 乙酰谷氨酸合酶（NAGS）催化。已知有不同种类的NAGS，例如“经典的”ArgA、双功能的ArgJ、ArgO和S - NAGS。然而，由于谷氨酸棒杆菌拥有单功能的ArgJ，其仅催化精氨酸生物合成途径的第五步，因此谷氨酸必须由一个尚未知的NAGS基因进行乙酰化。
通过使用预期会积累相应细胞内代谢物的特定基因缺失突变体进行代谢组分析来研究精氨酸生物合成。HPLC - ESI - qTOF分析通过检测精氨酸生物合成的七个中间产物中的六个，对精氨酸代谢有了详细的了解。所有突变体中N - 乙酰谷氨酸的积累进一步证实了未知的NAGS活性。为了阐明该基因的身份，构建了谷氨酸棒杆菌的基因组文库，并用于补充大肠杆菌ΔargA突变体。鉴定出的能够实现功能互补的质粒包含基因cg3035的一部分，该基因在其氨基酸序列中含有一个乙酰转移酶结构域。在谷氨酸棒杆菌基因组中缺失cg3035导致对精氨酸的部分营养缺陷。cg3035基因的异源过表达验证了其在体内补充大肠杆菌ΔargA突变体的能力，同源过表达导致细胞内N - 乙酰谷氨酸池显著增加。酶活性测定在体外证实了Cg3035的N - 乙酰谷氨酸合酶活性。然而，Cg3035的氨基酸序列与已知NAGS基因家族的成员没有相似性。
N - 乙酰谷氨酸合酶Cg3035能够催化谷氨酸棒杆菌中精氨酸生物合成的第一步。它代表了一类新的NAGS基因，显然仅存在于棒杆菌亚目的细菌中，其中包括谷氨酸棒杆菌属、分枝杆菌属和诺卡氏菌属等。因此，建议将这个新家族命名为C - NAGS（棒杆菌亚目型NAGS）。
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