F420H2 is required for phthiocerol dimycocerosates synthesis in mycobacteria.

Phthiocerol dimycocerosates (PDIM) are a group of cell surface-associated apolar lipids of Mycobacterium tuberculosis and closely related mycobacteria such as Mycobacterium bovis and Mycobacterium leprae A characteristic methoxy group of these lipids is generated from the methylation of a hydroxyl group of the direct precursors, the phthiotriols. The precursors arise from the reduction of phthiodiolones, the keto intermediates, by a ketoreductase. The putative phthiodiolone ketoreductase (PKR) is encoded by Rv2951c in M. tuberculosis and BCG_2972c in M. bovis BCG, and these ORFs have identical amino acid sequences. We have investigated the cofactor requirement of BCG_2972c. A comparative analysis based on the crystallographic structures of similar enzymes identified structural elements for binding coenzyme F420 and hydrophobic phthiodiolone in PKR. Coenzyme F420 is a deazaflavin coenzyme, serving several key functions in pathogenic and non-pathogenic mycobacteria. We found that a M. bovis BCG mutant lacking F420-dependent glucose-6-phosphate dehydrogenase (Fgd), which generates F420H2 (glucose-6-phosphate + F420 → 6-phosphogluconate + F420H2), was devoid of phthiocerols and accumulated phthiodiolones. When provided with F420H2, a broken cell slurry of the mutant converted accumulated phthiodiolones to phthiocerols; F420H2 was generated in situ from F420 and glucose-6-phosphate by the action of Fgd. Thus, the reaction mixture was competent in reducing phthiodiolones to phthiotriols (phthiodiolones + F420H2 → phthiotriols + F420), which was then methylated to phthiocerols. These results established the mycobacterial phthiodiolone ketoreductase as an F420H2-dependent enzyme (fPKR). A phylogenetic analysis of close homologs of fPKR revealed potential F420-dependendent lipid modifying enzymes in a broad range of mycobacteria.

IMPORTANCE: Mycobacterium tuberculosis is the causative agent of tuberculosis and phthiocerol dimycocerosates (PDIM) protect this pathogen from an early innate immune response of an infected host. Thus, the PDIM synthesis system is a potential target for the development of effective treatments for tuberculosis. The current study shows that a PDIM synthesis enzyme is dependent on coenzyme F420 F420 is universally present in mycobacteria and absent in humans. This finding expands the number of experimentally validated F420-dependent enzymes in M. tuberculosis to six, each of which helps the pathogen to evade killing by the host immune system and one activates an anti-tuberculosis drug, PA-824. This work is also of relevance to leprosy, since similar waxy lipids are found in Mycobacterium leprae.