title: Functional identification of MSMEG_6402 protein from Mycobacterium smegmatis in decaprenylphosphoryl-D-arabinose biosynthesis.
abstract: The arabinogalactan (AG) of the mycobacterial cell wall consists of an arabinan region, a galactan region and a disaccharide linker. Decaprenylphosphoryl-D-arabinose (DPA) is the donor for arabinofuran residues, which are formed from phosphoribose diphosphate (PRPP) and decaprenyl phosphate (DP). DP is sequentially catalyzed by a three-step process that involves a transferase, a phosphatase and an epimerase. Rv3807c is a putative phospholipid phosphatase that might generate the intermediate product of decaprenyl-phosphoryl-ribose (DPR) in DPA biosynthesis. Mycobacterium smegmatis MSMEG_6402 is a homolog gene of Mycobacterium tuberculosis Rv3807c and was substituted for the functional identification of Rv3807c. Previously, we generated a conditional MSMEG_6402 gene knockout strain (M. sm-ΔM_6402) that exhibited significantly affected cell wall structure. To understand the function of MSMEG_6402 in DPA biosynthesis, this gene was amplified and expressed, and the resulting protein was identified and purified using a His-tagged approach. A MSMEG_6402 enzymatic reaction system with PRPP and DP as substrates was utilized, and the reaction products were separated using thin layer chromatography (TLC). The results revealed a specific lipid-linked sugar band that appeared in the reaction with the addition of MSMEG_6402. Furthermore, ESI-MS detection was utilized in this study, and the results revealed that the enzymatic reaction products involving MSMEG_6402 included DPPR and a sodium ion adduct of DPR. Additionally, the phosphatase activity of MSMEG_6402 was also determined through phosphate group detection using the colorimetric method. Based on our results together with the results of previous studies, including the functional identification and bioinformatics analysis of M. tuberculosis Rv3807c, we propose that MSMEG_6402, as a phosphatase, has an intimate relationship with DPA biosynthesis.
authors.affiliation: Department of Biotechnology, Dalian Medical University, Dalian 116044, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Jiang
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044, China.
authors.forename: Lina
authors.initials: L
authors.lastname: Cai
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044, China.
authors.forename: Xiaojiao
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044, China.
authors.forename: Lianqi
authors.initials: L
authors.lastname: He
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044, China.
authors.forename: Yufang
authors.initials: Y
authors.lastname: Ma
authors.affiliation: Department of Biotechnology, Dalian Medical University, Dalian 116044, China.
authors.forename: Shizhu
authors.initials: S
authors.lastname: Zang
authors.affiliation: Department of Biotechnology, Dalian Medical University, Dalian 116044, China.
authors.forename: Cuili
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Biotechnology, Dalian Medical University, Dalian 116044, China.
authors.forename: Xinli
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Biotechnology, Dalian Medical University, Dalian 116044, China. Electronic address: jimxin@hotmail.com.
authors.forename: Yi
authors.initials: Y
authors.lastname: Xin
mesh_terms: D001089:Arabinose / Q000031:analogs & derivatives* / Q000096:biosynthesis; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics*; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D003124:Colorimetry; D017353:Gene Deletion; D015870:Gene Expression; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013729:Terpenes
chemical_list: D001426:Bacterial Proteins; D013729:Terpenes; C089328:decaprenyl-P-arabinose; D001089:Arabinose; D010744:Phosphoric Monoester Hydrolases
keywords: 5-Phosphoribose-1-diphosphate; Decaprenylphosphoryl-D-arabinose; MSMEG_6402; Mycobacterium smegmatis