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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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Decaprenylphosphoryl- d-arabinofuranosyl (DPA), the immediate donor for the polymerized d-Araf residues of mycobacterial arabinan, is synthesized from 5-phosphoribose-1-diphosphate (PRPP) in three-step reactions. (i) PRPP is transferred to decaprenyl-phosphate (DP) to form decaprenylphosphoryl- d-5-phosphoribose (DPPR). (ii) DPPR is dephosphorylated to form decaprenylphosphoryl- d-ribose (DPR). (iii) DPR is formed to DPA by the epimerase. M ycobacterium tuberculosis Rv3806c and heteromeric Rv3790/Rv3791 have been identified as the PRPP: decaprenyl-phosphate 5-phosphoribosyltransferase and the epimerase respectively. Rv3807c, however, as the candidate of phospholipid phosphatase, catalyzing the biosynthesis of decapreny-l-phosphoryl-ribose (DPR) from decaprenylphosphoryl-β- d-5-phosphoribose by dephosphorylating, has no direct experimental evidence of its essentiality in any species of mycobacterium. In this study, Rv3807c gene was amplified from the genome of M. tuberculosis H37Rv by PCR, and was successfully expressed in Escherichia coli BL21 (DE3) via the recombinant plasmid pColdII-Rv3807c. The resulting protein with the 6× His-tag was identified by SDS-PAGE and Western blotting. The protein was predicted through bioinformatics to contain three transmembrane domains, the N-terminal peptide, and a core structure with phosphatidic acid phosphatase type2/haloperoxidase. This study provides biochemical and bioinformatics evidence for the importance of Rv3807c in mycobacteria, and further functional studies will be conducted for validating Rv3807c as a promising phospholipid phosphatase in the synthetic pathway of DPA. [ABSTRACT FROM AUTHOR]