Huang Hairong, Berg Stefan, Spencer John S, Vereecke Danny, D'Haeze Wim, Holsters Marcelle, McNeil Michael R
Beijing Tuberculosis and Thoracic Tumor Institute, Beijing 101149, China.
Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
Microbiology (Reading). 2008 Mar;154(Pt 3):736-743. doi: 10.1099/mic.0.2007/013532-0.
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.
癸异戊二烯基磷酸 -D-阿拉伯糖（DPA）已被证明是结核分枝杆菌细胞壁中必需的D-阿拉伯呋喃糖残基的供体。DPA由磷酸核糖二磷酸通过一个四步过程形成。第一步是用癸异戊二烯基磷酸对二磷酸基团进行亲核取代。该反应由整合膜蛋白5-磷酸-α-D-核糖-1-二磷酸：癸异戊二烯基磷酸5-磷酸核糖基转移酶（DPPR合酶）催化。该酶对生长至关重要，因此是开发新型抗结核药物的重要潜在靶点。尽管膜蛋白是当前抗菌药物靶点的一个重要子集，但通过X射线晶体学通常不容易获得此类蛋白的结构和活性位点的详细信息。为了以不同方法解决这个问题，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，并显示为有活性的DPPR合酶。随后对排列好的序列进行了生物信息学分析，然后对被确定可能对活性重要的氨基酸进行定点诱变。结果表明，癸异戊二烯基磷酸5-磷酸核糖（DPPR）的酶促合成发生在质膜的细胞质一侧。氨基酸取代表明，预测的细胞质N端区域和两个细胞质环与一些相邻内膜区域的部分一起参与底物结合和/或催化。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，但含有酶活性所需的保守NDxxD基序。因此，这种DPPR合酶具有与经典真核酶不同的pRpp结合位点是合理的，从而鼓励进一步开展针对该酶开发抑制剂的工作。
