Abstract
BackgroundIt was proposed that there are at least 250 enzymes in M. tuberculosis involved in lipid metabolism. Rv0045c was predicted to be a hydrolase by amino acid sequence similarity, although its precise biochemical characterization and function remained to be defined.Methodology/Principal FindingsWe expressed the Rv0045c protein to high levels in E. coli and purified the protein to high purity. We confirmed that the prepared protein was the Rv0045c protein by mass spectrometry analysis. Circular dichroism spectroscopy analysis showed that the protein possessed abundant β-sheet secondary structure, and confirmed that its conformation was stable in the range pH 6.0–10.0 and at temperatures ≤40°C. Enzyme activity analysis indicated that the Rv0045c protein could efficiently hydrolyze short chain p-nitrophenyl esters (C2–C8), and its suitable substrate was p-nitrophenyl caproate (C6) with optimal catalytic conditions of 39°C and pH 8.0.Conclusions/SignificanceOur results demonstrated that the Rv0045c protein is a novel esterase. These experiments will be helpful in understanding ester/lipid metabolism related to M. tuberculosis.
Highlights
Mycobacterium tuberculosis (M. tuberculosis), firstly discovered by Robert Koch [1], is a pathogenic species and the causative agent of most tuberculosis [2]
In 1998, the whole genome of M. tuberculosis H37Rv strain was sequenced by the Sanger Center and the Institut Pasteur, showing at least 250 enzymes related to lipid metabolism including extracellular secreted enzymes, integrated cell wall enzymes and intracellular esterases/lipases, compared with about 50 enzymes in E. coli [6,7]
Esterases or lipases are types of hydrolases which are widely distributed from prokaryotes to eukaryotes, and which are involved in lipid metabolism
Summary
Mycobacterium tuberculosis (M. tuberculosis), firstly discovered by Robert Koch [1], is a pathogenic species and the causative agent of most tuberculosis [2]. M. tuberculosis has an unusual, waxy coating on the cell surface (primarily mycolic acid), which highlights that there must be a large number of enzymes involved in lipid metabolism. In 1998, the whole genome of M. tuberculosis H37Rv strain was sequenced by the Sanger Center and the Institut Pasteur, showing at least 250 enzymes related to lipid metabolism including extracellular secreted enzymes, integrated cell wall enzymes and intracellular esterases/lipases, compared with about 50 enzymes in E. coli [6,7]. The genomic organization and gene functionality of M. tuberculosis are invaluable for understanding the slowly growing pathogen. Mycobacterial genes that are involved in lipid metabolism, cell division chromosomal partitioning, and secretion are more likely to be required for survival in mice [8,9]. Lamichhane and colleagues detected 31 M. tuberculosis genes that were found to be required for in vivo survival in mouse lungs. Rv0045c was predicted to be a hydrolase by amino acid sequence similarity, its precise biochemical characterization and function remained to be defined
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