A novel type of N-acetylglutamate synthase is involved in the first step of arginine biosynthesis in Corynebacterium glutamicum.
A novel type of N-acetylglutamate synthase is involved in the first step of arginine biosynthesis in Corynebacterium glutamicum.
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DOI：
10.1186/1471-2164-14-713
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发表时间：
2013-10-18
期刊：
影响因子：
4.4
通讯作者：
Kalinowski J
中科院分区：
文献类型：
--
作者：
Petri K;Walter F;Persicke M;Rückert C;Kalinowski J
谷氨酸棒杆菌精氨酸的生物合成包括8个酶促步骤，首先是N-乙酰谷氨酸合成酶(NAGS)催化的谷氨酸乙酰化反应。已知的NAGS有不同的种类，例如，经典的ArgA、双功能的ArgJ、Argo和S-NAG。然而，由于谷氨酰胺具有一个单功能的ArgJ，它只催化精氨酸生物合成途径的第五步，因此谷氨酸必须被一个未知的NAGS基因乙酰化。精氨酸的生物合成通过代谢组谱进行研究，使用定义的基因缺失突变体，这些突变体预计会积累相应的细胞内代谢物。HPLC-ESI-QTOF分析通过检测精氨酸生物合成的七个中间体中的六个，对精氨酸代谢提供了详细的见解。N-乙酰谷氨酸在所有突变体中的积累进一步证实了未知的NAGS活性。为了阐明该基因的特性，我们建立了谷氨酸杆菌的基因组文库，并将其用于补充大肠杆菌Δarga突变体。鉴定出的能够进行功能互补的质粒含有cg3035基因的一部分，该基因的氨基酸序列中含有一个乙酰基转移酶结构域。谷氨酸杆菌基因组中cg3035的缺失导致了精氨酸的部分营养缺陷症。Cg3035基因的异源过表达在体内证实了其补充大肠杆菌Δarga突变体的能力，同源过表达导致细胞内N-乙酰谷氨酸库显著增加。酶活性测定证实Cg3035体外具有N-乙酰谷氨酸合成酶活性。然而，Cg3035的氨基酸序列与已知的NAGS基因家族成员没有相似性。N-乙酰谷氨酸合成酶Cg3035能够催化谷氨酸杆菌精氨酸生物合成的第一步。它代表了一类新的NAGS基因，显然只存在于棒状杆菌亚目的细菌中，其中包括棒状杆菌属、分枝杆菌属和诺卡氏菌属。因此，建议将这个新家族命名为C-NAGS(棒状杆菌亚科类型NAGS)。
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.
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影响因子：
3
作者：
Dondrup, Michael;Albaum, Stefan P.;Griebel, Thasso;Henckel, Kolja;Juenemann, Sebastian;Kahlke, Tim;Kleindt, Christiane K.;Kuester, Helge;Linke, Burkhard;Mertens, Dominik;Mittard-Runte, Virginie;Neuweger, Heiko;Runte, Kai J.;Tauch, Andreas;Tille, Felix;Puehler, Alfred;Goesmann, Alexander
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7.4
作者：
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通讯作者：
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2.8
作者：
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