Identification of amino acids and domains required for catalytic activity of DPPR synthase, a cell wall biosynthetic enzyme of Mycobacterium tuberculosis
Identification of amino acids and domains required for catalytic activity of DPPR synthase, a cell wall biosynthetic enzyme of Mycobacterium tuberculosis
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DOI：
10.1099/mic.0.2007/013532-0
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发表时间：
2008-03-01
期刊：
影响因子：
2.8
通讯作者：
McNeill, Michael R.
中科院分区：
文献类型：
--
作者：
Huang, Hairong;Berg, Stefan;McNeill, Michael R.
十异戊二烯磷酸-D-阿拉伯糖（DPA）已被证明是结核分枝杆菌细胞壁中发现的必需D-阿拉伯呋喃糖残基的供体。DPA是由磷酸核糖二磷酸在一个四步过程中形成的。第一步是用磷酸十异戊烯酯亲核取代二磷酸基团。该反应由整合膜蛋白5-磷酸-α-D-核糖-1-二磷酸：十异戊二烯基-磷酸5-磷酸核糖基转移酶（DPPR合成酶）催化。这种酶是生长所必需的，因此是开发新的结核病药物的重要目标候选者。虽然膜蛋白是目前抗菌剂的一个重要的靶点，但这些蛋白的结构和活性位点的细节通常不容易通过X射线晶体学获得。为了开始对该问题的不同方法，来自耻垢分枝杆菌和谷氨酸棒杆菌的同源物在大肠杆菌中表达，并显示为活性DPPR酶。随后对比对序列进行生物信息学分析，然后对鉴定为可能对活性重要的氨基酸进行定点诱变。结果表明，十异戊二烯磷酸5-磷酸核糖（DPPR）的酶促合成发生在质膜的细胞质侧。氨基酸取代表明，预测的细胞质N-末端区域和两个细胞质环参与底物结合和/或催化沿着与一些相邻的内膜区域的部分。该酶缺乏在原核生物和真核生物的核酸前体酶中发现的经典磷酸核糖二磷酸（pRpp）结合位点，而是含有酶活性所需的保守NDxxD基序。因此，这是合理的，这种DPPR合酶具有pRpp结合位点，这是不同于经典的真核生物酶，并进一步的工作，以开发针对这种酶的抑制剂，从而鼓励。
Decaprenylphosphoryl-D-arabinose (DPA) has been shown to be the donor of the essential D-arabinofuranosyl residues found in the cell wall of Mycobacterium tuberculosis. DPA is formed from phosphoribose diphosphate in a four-step process. The first step is the nucleophilic replacement of the diphosphate group with decaprenyl phosphate. This reaction is catalysed by the integral membrane protein 5-phospho-alpha-D-ribose-1-diphosphate: decaprenyl-phosphate 5-phosphoribosyltransferase (DPPR synthase). The enzyme is essential for growth and thereby an important target candidate for the development of new tuberculosis drugs. Although membrane proteins are an important subset of targets for current antibacterial agents, details about the structures and the active sites of such proteins are often not readily available by X-ray crystallography. To begin a different approach to the issue, homologues from Mycobacterium smegmatis and Corynebacterium glutamicum were expressed in Escherichia coli and shown to be active DPPR synthases. This was followed by bioinformatic analyses of the aligned sequences and then by site-directed mutagenesis of amino acids identified as likely to be important for activity. The results suggested that the enzymic synthesis of decaprenyl-phosphate 5-phosphoribose (DPPR) occurs on the cytoplasmic side of the plasma membrane. Amino acid substitutions showed that the predicted cytoplasmic N-terminal region and two cytoplasmic loops are involved in substrate binding and/or catalysis along with parts of some adjoining inner membrane regions. The enzyme lacks the classical phosphoribose diphosphate (pRpp) binding site found in nucleic acid precursor enzymes of both prokaryotes and eukaryotes but instead contains a conserved NDxxD motif required for enzymic activity. Thus, it is plausible that this DPPR synthase has a pRpp binding site that is different from that of the classical eukaryotic enzymes, and further work to develop inhibitors against this enzyme is thereby encouraged.