Article Figures & Data
Figures
- FIG 1
PEP, but not 2PL, stimulates DH-F420-0 synthesis in M. smegmatis cell lysates. (A) Two-dimensional (2D) structures of PEP and 2PL demonstrating the difference (double bond or single bond) in bonding between carbon 2 and 3. (B) Fluorescence emission detection chromatogram from HPLC of M. smegmatis lysates spiked with either 2PL or PEP or an unspiked control. Synthesis of a species with characteristic F420 fluorescence (excitation, 420 nm; emission, 480 nm) corresponding to F420-0 from the purified standard was detected only in the PEP-spiked lysate. The appearance of this F420-0-like species coincided with a decrease in the presence of Fo, suggesting that PEP is the precursor for F420 synthesis in M. smegmatis in cells. F420-1 in the standard corresponds to F420 with a single glutamate moiety.
- FIG 2
Mutagenic dissection of the F420 biosynthesis pathway in M. smegmatis reveals that DH-F420-0 is the biosynthetic intermediate in mycobacteria. (A) A schematic of the F420 biosynthesis pathway in M. smegmatis with PEP, rather than 2PL, utilized by FbiD to create the reaction intermediate EPPG. The enzymes responsible for catalytic steps are shown, along with the 2D structures of proposed pathway intermediates and mature F420. The yellow box highlights the reduction of DH-F420-0, proposed to be mediated by the C-terminal domain of FbiB using FMNH2. (B to D) LC-MS detection of mature F420 species (B), Fo (C), and DH-F420-0 (D) in M. smegmatis cell lysates of the wild type (Wt) and F420 biosynthesis pathway mutants confirming the proposed function of the F420 biosynthetic genes detecting the novel intermediate DH-F420-0 in whole cells. F420-X species in panel B correspond to different lengths of the polyglutamate chain where X = n tail length.
- FIG 3
The crystal structure of FbiA captures the enzyme in open and closed states. (A) The crystal structure of FbiA from M. smegmatis in complex with Fo and GDP. FbiA is shown as a cartoon representation with molecule B (Mol. B) in sky blue and Mol. A in light blue. GDP and Fo are shown as stick representations, and Ca2+ is shown as a yellow sphere. (B) Mol. A from the FbiA structure exists in an open conformation. (Left) Mol. A as a cartoon with loops and subdomains which differ in conformation in Mol. B highlighted in red. (Right) Mol. A as a surface representation with mobile regions highlighted in red. (C) Mol. B of FbiA structure exists in a closed “catalytically ready” state. (Left) Mol. B displayed as in panel B, with the direction of movement of loops compared to Mol. A shown with blue arrows. (Right) Mol. B as in panel B, demonstrating how the mobile regions enclose the FbiA active site.
- FIG 4
Resolution of the structure of FbiA in the presence of Fo, GDP, and DH-F420-0 provides insight into its catalytic mechanism. (A) Fo and GDP in complex with Mol. B of FbiA in coordination with the catalytic Ca2+ ion. FbiA is shown as a sky blue cartoon, Fo and GDP as sticks, and Ca2+ as a sphere. (B) Stereoview of the catalytic center of the FbiA active site in complex with Fo and GDP, showing FbiA side chains involved in coordinating the catalytic metal ion and a coordinating H2O molecule. Bond distances of <3.2 Å are shown as yellow dashed lines, and the distance between the terminal OH of Fo and P of the β-phosphate of GDP is highlighted in blue. (C) DH-F420-0 in complex with FbiA, shown as in panel A. (D) Stereoview of the FbiA catalytic center with the reaction substrate EPPG model in place of GDP displayed in panel C, with the close proximity between the carboxylic acid group of EPPG and the terminal OH of Fo highlighted with a red dashed line. (E) Schematic showing the proposed catalytic mechanism for the formation of DH-F420-0 by FbiA.
Supplemental Material
FIG S1
Fluorescence-coupled HPLC analysis of clarified cell lysates from M. smegmatis F420 biosynthesis pathway mutants. Fluorescence (excitation wavelength [Ex λ] of 420 nm; emission wavelength [Em λ] of 480 nm) trace for wild-type (A), ΔfbiC (B), ΔfbiA (C), ΔfbiD (D), and ΔfbiB (E) strains showing the formation of mature F420 species in the wild-type strain only and accumulation of DH-F420-0 in the ΔfbiB strain. LU, fluorescence intensity. For ΔfbiC, shown in panel B, mass spectrometry analysis confirmed that no Fo or F420 species were present, despite observed low-level fluorescence. Download FIG S1, TIF file, 1.4 MB.
Copyright © 2020 Grinter et al.
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FIG S2
FbiA recombinantly expressed in M. smegmatis copurifies with its product DH-F420-0. (A) Purified, concentrated (16 mg · ml−1) recombinant FbiA produced in M. smegmatis showing a characteristic yellow color associated with a bound F420 species. (B) The absorbance (purple) and fluorescence spectra (blue) of purified FbiA from panel A, which is characteristic of F420 species with a protonated deazaflavin 8-OH group. (C) LC-MS analysis of F420 species bound to purified FbiA, showing that DH-F420-0 is the predominantly bound species and the presence of some mature F420 species. (D) SEC-MALS analysis of purified FbiA shows that it has a molecular weight in solution consistent with a homodimeric species. Download FIG S2, TIF file, 2.3 MB.
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FIG S3
Electron density corresponding to FbiA substrates and products in cocrystal structures. The panel corresponds to the cocrystal structures indicated in the bottom right. A composite omit map is shown carved to visible molecules at a distance of 2 Å and contoured to 1 σ. Download FIG S3, TIF file, 1.8 MB.
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TABLE S1
Crystallographic data collections and refinement statistics. Download Table S1, XLSX file, 0.01 MB.
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TABLE S2
FbiA dimer interface statistics reported by PISA (29). Download Table S2, XLSX file, 0.01 MB.
Copyright © 2020 Grinter et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S4
The crystal structure of FbiA in complex with Fo and GDP. (A) GDP in complex with Mol. B of FbiA. FbiA is represented as a sky blue cartoon, bound GDP and glycerol molecules are shown as stick models, and bound Ca2+ ion is shown as a yellow sphere. (B) A stereoview of the catalytic center of the FbiA active site in complex with GDP and glycerol, showing FbiA side chains involved in coordinating the catalytic metal ion and a coordinating H2O molecule. Bond distances of <3.2 Å are shown as yellow dashed lines. (C) Fo in complex with Mol. B of FbiA is shown as a sky blue cartoon, and Fo is shown as a stick model. Download FIG S4, TIF file, 2.7 MB.
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FIG S5
The full interaction network of the FbiA active site in the presence of substrates (Fo and GDP) and product (DH-F420-0). Download FIG S5, TIF file, 2.0 MB.
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TABLE S3
Oligonucleotide primers utilized in this study. Download Table S3, XLSX file, 0.01 MB.
Copyright © 2020 Grinter et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
