A revised biosynthetic pathway for the cofactor F420 in prokaryotes

Bashiri, Ghader; Antoney, James; Jirgis, E.N.M.; Shah, Mihir V.; Ney, Blair; Copp, Janine N.; Stuteley, Stephanie M.; Sreebhavan, Sreevalsan; Palmer, Brian D.; Middleditch, Martin; Tokuriki, Nobuhiko; Greening, Chris; Scott, Colin; Baker, Edward N.; Jackson, Colin J.

doi:10.1038/s41467-019-09534-x

Cited by 63 publications

(159 citation statements)

References 69 publications

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“…Surprisingly, Mtb has four close homologs of BVR even though Mtb does not have a conventional BV-producing HO enzyme (Chim et al, 2010). One of these homologs, Rv2074, has BV reduction activity although its electron donating cofactor is the flavin cofactor F420 (Ahmed et al, 2016), a deazaflavin cofactor that is a low potential hydride transfer agent (Bashiri et al, 2019), rather than flavin mononucleotide (FMN) as observed for eukaryotic BVRs (Sugishima et al, 2018). In contrast, Rv1155…”

Section: Discussionmentioning

confidence: 99%

The structure ofMycobacterium tuberculosisheme-degrading protein, MhuD, in complex with product

Chao

Burley

Sieminski

et al. 2019

Preprint

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Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, requires iron for survival. In Mtb, MhuD is the cytosolic protein that degrades imported heme. MhuD is distinct, both in sequence and structure, from canonical heme oxygenases (HOs) but homologous with IsdG-type proteins. Canonical HO is found mainly in eukaryotes, while IsdG-type proteins are predominantly found in prokaryotes including pathogens. While there are several published structures of MhuD and other IsdG-type proteins in complex with heme substrate, no structures have been reported of IsdG-type proteins in complex with product, unlike HOs. We recently showed that the Mtb variant MhuD-R26S produces biliverdin IXa (aBV) rather than the wild-type (WT) mycobilin isomers as product. Given that mycobilin and other IsdG-type protein products like staphylobilin are difficult to isolate in quantities sufficient for structure determination, here we use the MhuD-R26S variant and its product aBV as a proxy to study the IsdG-type protein/product complex. First we show that aBV has nanomolar affinity for MhuD and the R26S variant. Second we determined the MhuD-R26S-aBV complex structure to 2.5 Å, which reveals two notable features (1) two aBV molecules bound per active site and (2) a new a-helix (a3) as compared with the MhuD-heme structure. Finally, by molecular dynamics simulations we show that a3 is stable with the proximal aBV alone. MhuD's high affinity for its product and structural and electrostatic changes that accompany substrate turnover suggest that there is an unidentified protein that is responsible for product extraction from MhuD and other IsdG-type proteins.

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Section: Discussionmentioning

confidence: 99%

The structure ofMycobacterium tuberculosisheme-degrading protein, MhuD, in complex with product

Chao

Burley

Sieminski

et al. 2019

Preprint

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“…It was assumed that the biosynthesis pathway for archaeal F 420 was generic to all F 420 producing organisms (1). However, recent studies have shown that the structure and biosynthesis of F 420 varies between producing organisms (24, 26). F 420 produced by the proteobacterial fungal symbiont Paraburkholderia rhizoxinica was found to incorporate 3-phospho-D-glycerate (3PG) in the place of 2PL, producing a chemically distinct F 420 (26).…”

Section: Introductionmentioning

confidence: 99%

“…F 420 produced by the proteobacterial fungal symbiont Paraburkholderia rhizoxinica was found to incorporate 3-phospho-D-glycerate (3PG) in the place of 2PL, producing a chemically distinct F 420 (26). In parallel, analysis of purified F 420 biosynthesis enzymes from mycobacteria indicated that the central glycolytic and gluconeogenic intermediate phosphoenolpyruvate (PEP), rather than 2PL, is a precursor for F 420 biosynthesis (24). In contrast to P. rhizoxinica , in mycobacteria, mature F 420 is chemically analogous to that produced by archaea (27).…”

Section: Introductionmentioning

confidence: 99%

“…However, as these enzymes catalyze reactions distinct to their archaeal homologues, the following alternative nomenclature is applied compared to the archaeal enzymes: FbiD (homologous to CofC), FbiC (a single protein with domains homologous to CofG and CofH), FbiA (homologous to CofD) and FbiB (N-terminal domain homologous to CofE) (8). In addition to its CofE-like domain, FbiB possesses an FMN-binding C-terminal domain and biochemical evidence suggests it is responsible for the reduction of the moiety derived from PEP (24, 28).…”

Section: Introductionmentioning

confidence: 99%

“…The predicted use of PEP in F 420 biosynthesis in mycobacteria would lead to the formation of the oxidized intermediate compound dehydro-F 420 -0 (DH-F 420 -0). The production of DH-F 420 -0 was detected in a coupled enzyme assay containing purified FbiD and FbiA with PEP supplied as the substrate but has yet to be detected in mycobacterial cells (24). The study also showed that CofC from Methanocaldococcus jannaschii utilized PEP rather than 2PL for F 420 biosynthesis (24), leading the authors to conclude that PEP is the general precursor for F 420 biosynthesis in prokaryotes.…”

Section: Introductionmentioning

confidence: 99%

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

Grinter

Ney

Brammananth

et al. 2020

Preprint

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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...

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Natural Flavins: Occurrence, Role, and Noncanonical Chemistry

Drenth¹,

Fraaije²

2021

Flavin‐Based Catalysis

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A revised biosynthetic pathway for the cofactor F420 in prokaryotes

Cited by 63 publications

References 69 publications

The structure ofMycobacterium tuberculosisheme-degrading protein, MhuD, in complex with product

The structure ofMycobacterium tuberculosisheme-degrading protein, MhuD, in complex with product

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

Natural Flavins: Occurrence, Role, and Noncanonical Chemistry

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A revised biosynthetic pathway for the cofactor F420 in prokaryotes

Cited by 63 publications

References 69 publications

The structure ofMycobacterium tuberculosisheme-degrading protein, MhuD, in complex with product

The structure ofMycobacterium tuberculosisheme-degrading protein, MhuD, in complex with product

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

Natural Flavins: Occurrence, Role, and Noncanonical Chemistry

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Product

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