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Published on: April 25, 2014
Prokaryotic Expression, Identification and Bioinformatics Analysis of the Mycobacterium tuberculosis Rv3807c Gene
Lina Cai1, Xiaojiao Zhao2, Tao Jiang1
1Department of Biotechnology, Dalian Medical University, Dalian, 116044 People's Republic of China.
Abstract:
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. Mycobacterium 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.
Insights
This study investigates Rv3807c
Area of Science:
- Mycobacterial cell wall biosynthesis
- Enzymology
- Molecular biology
Background:
- Decaprenylphosphoryl-d-arabinofuranosyl (DPA) is crucial for mycobacterial arabinan synthesis.
- The enzyme Rv3807c is a candidate phospholipid phosphatase in the DPA pathway.
- Experimental evidence for Rv3807c's essentiality in mycobacteria is lacking.
Purpose of the Study:
- To biochemically and bioinformatically characterize Rv3807c.
- To provide evidence for Rv3807c's role in DPA biosynthesis.
- To validate Rv3807c as a potential drug target.
Main Methods:
- Gene amplification of Rv3807c from M. tuberculosis H37Rv.
- Recombinant expression of Rv3807c in E. coli.
- Protein identification using SDS-PAGE and Western blotting.
- Bioinformatic analysis of Rv3807c structure and function.
Main Results:
- Rv3807c was successfully expressed and purified.
- Bioinformatics predicted transmembrane domains and a catalytic core.
- Biochemical and bioinformatics data support Rv3807c's role in DPA synthesis.
Conclusions:
- Rv3807c is important for mycobacteria.
- Rv3807c functions as a phospholipid phosphatase in DPA synthesis.
- Rv3807c is a promising target for further functional studies and drug development.
