Abstract
ABSTRACTPhosphopantetheinyl hydrolase, PptH (Rv2795c), is a recently discovered enzyme from Mycobacterium tuberculosis that removes 4′-phosphopantetheine (Ppt) from holo-carrier proteins (CPs) and thereby opposes the action of phosphopantetheinyl transferases (PPTases). PptH is the first structurally characterized enzyme of the phosphopantetheinyl hydrolase family. However, conditions for optimal activity of PptH have not been defined, and only one substrate has been identified. Here, we provide biochemical characterization of PptH and demonstrate that the enzyme hydrolyzes Ppt in vitro from more than one M. tuberculosis holo-CP as well as holo-CPs from other organisms. PptH provided the only detectable activity in mycobacterial lysates that dephosphopantetheinylated acyl carrier protein M (AcpM), suggesting that PptH is the main Ppt hydrolase in M. tuberculosis. We could not detect a role for PptH in coenzyme A (CoA) salvage, and PptH was not required for virulence of M. tuberculosis during infection of mice. It remains to be determined why mycobacteria conserve a broadly acting phosphohydrolase that removes the Ppt prosthetic group from essential CPs. We speculate that the enzyme is critical for aspects of the life cycle of M. tuberculosis that are not routinely modeled.IMPORTANCE Tuberculosis (TB), caused by Mycobacterium tuberculosis, was the leading cause of death from an infectious disease before COVID, yet the in vivo essentiality and function of many of the protein-encoding genes expressed by M. tuberculosis are not known. We biochemically characterize M. tuberculosis’s phosphopantetheinyl hydrolase, PptH, a protein unique to mycobacteria that removes an essential posttranslational modification on proteins involved in synthesis of lipids important for the bacterium’s cell wall and virulence. We demonstrate that the enzyme has broad substrate specificity, but it does not appear to have a role in coenzyme A (CoA) salvage or virulence in a mouse model of TB.
Highlights
Phosphopantetheinyl hydrolase, phosphopantetheinyl hydrolase (PptH) (Rv2795c), is a recently discovered enzyme from Mycobacterium tuberculosis that removes 49-phosphopantetheine (Ppt) from holo-carrier proteins (CPs) and thereby opposes the action of phosphopantetheinyl transferases (PPTases)
Holo-acyl carrier protein M (AcpM) shifted to apo-AcpM in whole-cell lysates from wild-type bacteria but was stable in lysates lacking PptH or containing the inactive H246N mutant of PptH (7) (Fig. 4). These results suggest that PptH is the only Ppt hydrolase in M. tuberculosis that can remove Ppt from holo-AcpM under the conditions studied
The crystal structure of PptH placed it in a new family of phosphohydrolases and revealed an active site occupied by a Mn-Fe binuclear center (8)
Summary
Phosphopantetheinyl hydrolase, PptH (Rv2795c), is a recently discovered enzyme from Mycobacterium tuberculosis that removes 49-phosphopantetheine (Ppt) from holo-carrier proteins (CPs) and thereby opposes the action of phosphopantetheinyl transferases (PPTases). We could not detect a role for PptH in coenzyme A (CoA) salvage, and PptH was not required for virulence of M. tuberculosis during infection of mice It remains to be determined why mycobacteria conserve a broadly acting phosphohydrolase that removes the Ppt prosthetic group from essential CPs. We speculate that the enzyme is critical for aspects of the life cycle of M. tuberculosis that are not routinely modeled. M. tuberculosis AcpS and PptT have distinct substrates; AcpS substrates include FAS I and FAS II ACPs, whereas PptT substrates include PKS ACP and NRPS PCP Together, these two PPTases are involved in the production of unusual mycobacterial lipids that function in both cell wall structure and virulence (14). In contrast to AcpS, PptT is essential for the survival of M. tuberculosis in vitro and in vivo (15)
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