Taylor Zane W, Chamberlain Alexandra R, Raushel Frank M
Department of Biochemistry and Biophysics , Texas A&M University , College Station , Texas 77843 , United States.
Department of Chemistry , Texas A&M University , College Station , Texas 77843 , United States.
Biochemistry. 2018 Sep 18;57(37):5447-5455. doi: 10.1021/acs.biochem.8b00811. Epub 2018 Sep 6.
Campylobacter jejuni, a leading cause of gastroenteritis worldwide, has a unique O-methyl phosphoramidate (MeOPN) moiety attached to its capsular polysaccharide. Investigations into the biological role of MeOPN have revealed that it contributes to the pathogenicity of C. jejuni, and this modification is important for the colonization of C. jejuni. Previously, the reactions catalyzed by four enzymes (Cj1418-Cj1415) from C. jejuni that are required for the biosynthesis of the phosphoramidate modification have been elucidated. Cj1418 (l-glutamine kinase) catalyzes the formation of the initial phosphoramidate bond with the ATP-dependent phosphorylation of the amide nitrogen of l-glutamine. Here we show that Cj1418 catalyzes the phosphorylation of l-glutamine through a three-step reaction mechanism via the formation of covalent pyrophosphorylated ( Enz-X-P-P) and phosphorylated ( Enz-X-P) intermediates. In the absence of l-glutamine, the enzyme was shown to catalyze a positional isotope exchange (PIX) reaction within β-[O]-ATP in support of the formation of the Enz-X-P-Pintermediate. In the absence of ATP, the enzyme was shown to catalyze a molecular isotope exchange (MIX) reaction between l-glutamine phosphate and [N-amide]-l-glutamine in direct support of the Enz-X-Pintermediate. The active site nucleophile has been identified as His-737 based on the lack of activity of the H737N mutant and amino acid sequence comparisons. The enzyme was shown to also catalyze the phosphorylation of d-glutamine, γ-l-glutamyl hydroxamate, γ-l-glutamyl hydrazide, and β-l-aspartyl hydroxamate, in addition to l-glutamine.
空肠弯曲菌是全球肠胃炎的主要病因，其荚膜多糖上连接有独特的O - 甲基磷酰胺（MeOPN）部分。对MeOPN生物学作用的研究表明，它有助于空肠弯曲菌的致病性，并且这种修饰对于空肠弯曲菌的定殖很重要。此前，已经阐明了空肠弯曲菌中四种酶（Cj1418 - Cj1415）催化磷酰胺修饰生物合成所需的反应。Cj1418（L - 谷氨酰胺激酶）通过L - 谷氨酰胺酰胺氮的ATP依赖性磷酸化催化初始磷酰胺键的形成。在这里，我们表明Cj1418通过形成共价焦磷酸化（Enz - X - P - P）和磷酸化（Enz - X - P）中间体的三步反应机制催化L - 谷氨酰胺的磷酸化。在没有L - 谷氨酰胺的情况下，该酶被证明能催化β - [O] - ATP内的位置同位素交换（PIX）反应，以支持Enz - X - P - P中间体的形成。在没有ATP的情况下，该酶被证明能催化L - 谷氨酰胺磷酸与[N - 酰胺] - L - 谷氨酰胺之间的分子同位素交换（MIX）反应，直接支持Enz - X - P中间体。基于H737N突变体缺乏活性和氨基酸序列比较，已确定活性位点亲核试剂为His - 737。除了L - 谷氨酰胺外，该酶还被证明能催化D - 谷氨酰胺、γ - L - 谷氨酰羟肟酸、γ - L - 谷氨酰肼和β - L - 天冬氨酰羟肟酸的磷酸化。
