Characterization of Phosphopantetheinyl Hydrolase from Mycobacterium tuberculosis.

Phosphopantetheinyl 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.

Structural insights into phosphopantetheinyl hydrolase PptH from Mycobacterium tuberculosis.

The amidinourea 8918 was recently reported to inhibit the type II phosphopantetheinyl transferase (PPTase) of Mycobacterium tuberculosis (Mtb), PptT, a potential drug-target that activates synthases and synthetases involved in cell wall biosynthesis and secondary metabolism. Surprisingly, high-level resistance to 8918 occurred in Mtb harboring mutations within the gene adjacent to pptT, rv2795c, highlighting the role of the encoded protein as a potentiator of the bactericidal action of the amidinourea. Those studies revealed that Rv2795c (PptH) is a phosphopantetheinyl (PpT) hydrolase, possessing activity antagonistic with respect to PptT. We have solved the crystal structure of Mtb's phosphopantetheinyl hydrolase, making it the first phosphopantetheinyl (carrier protein) hydrolase structurally characterized. The 2.5 Å structure revealed the hydrolases' four-layer (α/ß/ß/α) sandwich fold featuring a Mn-Fe binuclear center within the active site. A structural similarity search confirmed that PptH most closely resembles previously characterized metallophosphoesterases (MPEs), particularly within the vicinity of the active site, suggesting that it may utilize a similar catalytic mechanism. In addition, analysis of the structure has allowed for the rationalization of the previously reported PptH mutations associated with 8918-resistance. Notably, differences in the sequences and predicted structural characteristics of the PpT hydrolases PptH of Mtb and E. coli's acyl carrier protein hydrolase (AcpH) indicate that the two enzymes evolved convergently and therefore are representative of two distinct PpT hydrolase families.