Cellular and structural basis of synthesis of the unique intermediate dehydro-F₄₂₀-0 in mycobacteria

Abstract

28F420 is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, 29 this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and 30 activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent 31 biochemical study proposed a revised biosynthesis pathway for F420 in mycobacteria; it was 32 suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather 33 than 2-phospholactate as long proposed, but these findings were subsequently challenged. In 34 this work, we combined metabolomic, genetic, and structural analyses to resolve these 35 discrepancies and determine the basis of F420 biosynthesis in mycobacterial cells. We show that, 36 in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-37 phospholactate stimulates F420 biosynthesis. Analysis of F420 biosynthesis intermediates 38 present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic 39 pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor 40 compound dehydro-F420-0. To determine the structural basis of dehydro-F420-0 production, we 41 solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo, substrate, 42 and product bound forms. These data show the essential role of a single divalent cation in 43 coordinating the catalytic pre-complex of this enzyme and demonstrate that dehydro-F420-0 44 synthesis occurs through a direct substrate transfer mechanism. Together, these findings 45 resolve the biosynthetic pathway of F420 in mycobacteria and have significant implications for 46 understanding the emergence of antitubercular prodrug resistance. 47 48 49 50 51 52 53 54 55 56Factor 420 (F420) is a deazaflavin cofactor that mediates diverse redox reactions in bacteria and 57 archaea (1). Chemically, F420 consists of a redox-active deazaflavin headgroup (derived from 58 the chromophore Fo) that is conjugated to a variable-length polyglutamate tail via a 59 phosphoester linkage (2). While the Fo headgroup of F420 superficially resembles flavins (e.g. 60FAD, FMN), three chemical substitutions in the isoalloxazine ring give it distinct chemical 61 properties more reminiscent of nicotinamides (e.g. NADH, NADPH) (1). These include a low 62 standard potential (-350 mV) and obligate two-electron (hydride) transfer chemistry (3, 4). The 63 electrochemical properties of F420 make it ideal to reduce a wide range of otherwise 64 recalcitrant organic compounds (5-7). Diverse prokaryotes are known to synthesize F420, but 65 the compound is best characterised for its roles in methanogenesis in archaea, antibiotic 66 biosynthesis in streptomycetes, and metabolic adaptation of mycobacteria (1,(8)(9)(10)(11). In 67 mycobacteria, F420 is involved in a plethora of processes: central carbon metabolism, cell wall 68 synthesis, recovery from dormancy, resistance to oxidative stress, and inactivation of certain 69 bactericidal agents (7,(12)(13)(1...

Mycobacterium tuberculosis complex lineage 5 exhibits high levels of within-lineage genomic diversity and differing gene content compared to the type strain H37Rv

“…Vitamin B6 is essential for the survival and virulence of M. tuberculosis [ 68 ] and its cofactor (F420-dependent biliverdin reductase) is implicated in immune-evasive mechanisms to allow bacterial persistence [ 66, 67 ] (Table S3). Recently, it has been reported that the synthesis of F420 might be stimulated by phosphoenolpyruvate [ 69 ], which is the precursor to pyruvate, a supplement often added to culture medium to improve the in vitro growth of L5, L6 and M. bovis strains. Furthermore, there is a suggestion that F420 is needed for the activation of the antituberculosis drugs pretomanid and delamanid [ 69 ].…”

“…Recently, it has been reported that the synthesis of F420 might be stimulated by phosphoenolpyruvate [ 69 ], which is the precursor to pyruvate, a supplement often added to culture medium to improve the in vitro growth of L5, L6 and M. bovis strains. Furthermore, there is a suggestion that F420 is needed for the activation of the antituberculosis drugs pretomanid and delamanid [ 69 ].…”

Modulation of riboflavin biosynthesis and utilization in mycobacteria

“…All analyses for these were performed using GraphPad Prism 9 with a statistical significance established at p <0.05. FbiC, FbiB and FbiA, 5-A-RU can also be converted to F420 (62). DMRL and its derivatives can act as MAIT cell activators.…”

Mycobacterium tuberculosiscomplex lineage 5 exhibits high levels of within-lineage genomic diversity and differing gene content compared to the type strain H37Rv

“…Vitamin B6 is essential for survival and virulence of M. tuberculosis (Dick et al 2010) and its cofactor (F420 dependent biliverdin reductase) is implicated in immune-evasive mechanisms to allow bacterial persistence (Ahmed et al 2016;Selengut and Haft 2010) (Table S6). Recently, it has been reported that the synthesis of F420 might be stimulated by phosphoenolpyruvate (Grinter et al 2020), which is the precursor to pyruvate, a supplement often added to culture medium to improve the in vitro growth of L5, L6 and M. bovis. Furthermore, there is a suggestion that F420 is needed for the activation of the antituberculosis drugs pretomanid and delamanid (Grinter et al 2020).…”

“…Recently, it has been reported that the synthesis of F420 might be stimulated by phosphoenolpyruvate (Grinter et al 2020), which is the precursor to pyruvate, a supplement often added to culture medium to improve the in vitro growth of L5, L6 and M. bovis. Furthermore, there is a suggestion that F420 is needed for the activation of the antituberculosis drugs pretomanid and delamanid (Grinter et al 2020).…”