UbiX is a flavin prenyltransferase required for bacterial ubiquinone biosynthesis

Abstract

Ubiquinone (also known as coenzyme Q) is a ubiquitous lipid-soluble redox cofactor that is an essential component of electron transfer chains. Eleven genes have been implicated in bacterial ubiquinone biosynthesis, including ubiX and ubiD, which are responsible for decarboxylation of the 3-octaprenyl-4-hydroxybenzoate precursor. Despite structural and biochemical characterization of UbiX as a flavin mononucleotide (FMN)-binding protein, no decarboxylase activity has been detected. Here we report that UbiX produces a novel flavin-derived cofactor required for the decarboxylase activity of UbiD. UbiX acts as a flavin prenyltransferase, linking a dimethylallyl moiety to the flavin N5 and C6 atoms. This adds a fourth non-aromatic ring to the flavin isoalloxazine group. In contrast to other prenyltransferases, UbiX is metal-independent and requires dimethylallyl-monophosphate as substrate. Kinetic crystallography reveals that the prenyltransferase mechanism of UbiX resembles that of the terpene synthases. The active site environment is dominated by π systems, which assist phosphate-C1' bond breakage following FMN reduction, leading to formation of the N5-C1' bond. UbiX then acts as a chaperone for adduct reorientation, via transient carbocation species, leading ultimately to formation of the dimethylallyl C3'-C6 bond. Our findings establish the mechanism for formation of a new flavin-derived cofactor, extending both flavin and terpenoid biochemical repertoires.

Extended Data Figure 1

a: Structural elucidation of the reduced UbiX/Fdc1 co-factor. From an initial full scan TIC on UbiX/Fdc1 extract, a 525 m/z ion extracted chromatogram was created under a gradient elution using H₂O/ acetonitrile both containing 0.1% Formic Acid indicating a major peak apex at 9.57 mins with a 54/46 solvent elution composition (not shown). Subsequent data dependant TIC and 525 m/z scan extracted chromatograms were created under 55% A / 45% B isocratic solvent elution and ion extraction between 524.5-525.5 m/z produced a singular peak at 2.28mins displaying an associated full scan molecular ion peak with m/z = 525.1726 (M⁺ = C₂₂H₃₀N₄O₉P) at a resolution of 58,500 with a mass accuracy of 3.59 ppm. Fragmentation of the 525.1726 m/z molecular ion peak in an automated data dependent manner using helium based-chemical induced dissociation (CID level 35) generated a spectral tree that indicates the removal of the newly formed, more labile, tertiary ring at the MS² level. Subsequent removal of the phosphate head group at the MS³ level was achieved using CID 35 on the 456.23 m/z molecular species with a final MS⁴ step using CID 35 on 358.18 m/z completely removing the tail group from the central 3-ring isoalloxazine system. b: Structural elucidation of the re-oxidised UbiX/Fdc1 co-factor. From an initial full scan TIC on UbiX/Fdc1 extract (i), a 524 m/z ion extracted chromatogram was created under a gradient elution using H₂O/ acetonitrile both containing 0.1% Formic Acid indicating a major peak apex at 9.24 mins with a 48/52 solvent composition (not shown). Subsequent data dependant TIC and 524 m/z scan extracted chromatograms (ii) were created under 50% A / 50% B isocratic solvent elution and ion extraction between 523.5-524.5 m/z produced a singular peak at 2.08mins displaying an associated full scan molecular ion peak with m/z = 524.1656 (M⁺ = C₂₂H₂₉N₄O₉P) at a resolution of 58,500 with a mass accuracy of 2.78 ppm. Fragmentation of the 524.1656 m/z molecular ion peak in an automated data dependent manner using helium based-chemical induced dissociation (CID level 35) generated a spectral tree (iii) that indicates the removal of the newly formed, more labile, tertiary ring at the MS² level. Subsequent removal of the phosphate head group at the MS³ level was achieved using CID 35 on the 498.31 m/z molecular species to create 327.18 (A) alongside a sister fragment 455.31 (B) that represents the full removal of the tertiary ring but retaining the phosphate head group.

Extended data Figure 2

Panel A. X-band continuous wave EPR spectra of UbiX in frozen solution: i) WT as isolated; ii) WT plus DMAP; iii) WT reduced with dithionite; iv) WT + DMAP reduced with dithionite; v) WT + DMAP reduced with dithionite and reoxidised with oxygen; vi) Y169F mutant + DMAP reduced with dithionite and reoxidised with oxygen; vii) W200F mutant + DMAP reduced with dithionite and reoxidised with oxygen. Clearly the FMN-DMAP adduct radical is only formed when UbiX is reoxidised in the presence of DMAP and this formation is not affected by mutation of those aromatic residues forming the π-cage that could give rise to Y or W radical species. Panel B. X-band continuous wave EPR spectra of frozen solutions of WT UbiX + DMAP and reduced with dithionite with the addition of potassium ferricyanide to the following concentrations: i) 260 μM; ii) 160 μM; iii) 50 μM; iv) 40 μM; v) 30 μM; vi) 20 μM; vii) 0 μM. Experimental conditions: microwave power 10 μW, modulation amplitude 1.5 G, temperature 20 K. Showing the radical can also be formed using chemical oxidation in the absence of oxygen and thus does not arise from a peroxide species generated by the reaction of reduced oxygen species formed when the dithionite sample is exposed to oxygen. An initial radical is formed under these conditions that exhibits a considerably broader EPR signal than the prFMN^radical and is as yet unidentified.

Extended Data Figure 3

Pulsed Davies ENDOR spectra of the prFMN^radical:UbiX complex. The spectrum was measured at a field equivalent to g_av = 2.0033. While a complete assignment of the spectrum requires specific deuteration of FMN and DMAP, the ENDOR spectrum is dominated by two large hyperfine couplings to β-protons indicated as H_A and H_B. Using the Heller-McConnell equation the values of the dihedral angles, θ, can be determined as shown and are consistent with the orientation of the C1’-protons of the DMAP-derived fragment of the radical observed crystallographically, as shown in the figure above. The unpaired electron spin density, ρ, at N5 of the FMN-derived fragment of the radical can also be estimated from the Heller-McConnell equation. B’ is negligible while B” is thought to have a value of ~160, although studies of β-protons coupled to unpaired electron spin density at a nitrogen atom are rare, giving an unpaired spin density at N5 of ~0.3, consistent with calculations and considerably smaller than the unpaired electron spin density of 0.4 or greater expected for C1’ of an aromatic amino acid radical.

Extended Data Figure 4

Top, DFT model of the purple radical species showing the location of significant atomic spin densities (>|0.02|) to the right. The optimised structure (blue carbons) overlaid with the crystal coordinates (green carbons) is shown below. The model was geometry optimised in the gas phase using the UB3LYP/6-311++G(d,p) level of theory. Cartesian coordinates of the optimised structure are given in Supplementary Information.

Extended Data Figure 5

A) Reconstitution of A. niger Fdc activity with UbiX:prFMN^reduced and prFMN^reduced obtained through filtration of a UbiX:prFMN^reduced reaction. Control reactions are devoid of any DMAP substrate. B) Rate of formation of spectral species 2 (see Fig 1f) in function of DMAP concentration. C) Rate of decay of spectral species 2 (see Fig 1f) in function of DMAP concentration. D) Spectral species obtained from singular value decomposition of rapid-scan stopped-flow spectrophotometric data following mixing of UbiX:prFMN^reduced with oxygenated buffer. E) The rate of purple radical (species B in panel d of this figure) formation as obtained from singular value decomposition of rapid-scan stopped-flow spectrophotometric data following mixing of UbiX:prFMN^reduced with oxygenated buffer has a linear dependence on oxygen concentration. Error bars are s.e.m. n=3

Extended Data Figure 6

Multiple sequence alignment of UbiX/Pad enzymes from selected bacterial or fungal species. Pseudomonas aeruginosa UbiX (NP_252708), Escherichia coli O157:H7 EcdB (NP_311620), Escherichia coli UbiX (YP_490553), Bacillus subtilis BsdB (WP_009966530), Saccharomyces cerevisiae Pad1 (AAB64980), Aspergillus niger PadA1 (ABN13117), and orf8 from the Thauera aromatica phenylphosphate carboxylase gene cluster (PAAD_THAAR). Conserved residues involved in phosphate binding, N5 polar network or formation of the substrate binding p-cage are indicated by labelled arrows. Secondary structure elements of P. aeruginosa UbiX crystal structure are shown. Alpha-helices and 3₁₀-helices (denoted as n) are shown as squiggles, β-strands by arrows and β-turns as TT.

Extended Data Figure 7

Crystal structure of P. aeruginosa UbiX:FMN:DMAP flash cooled to 100K at 30 s following complete reduction by sodium dithionite. Two orientations are displayed as in Fig 2. The omit map for the prFMN^reduced product is shown as green mesh, contoured at 4 sigma.

Extended Data Figure 8

Crystal structures of P. aeruginosa UbiX^Y169F a) Detailed view of the UbiX^Y169:FMN:DMAP complex with individual amino acids contributing to active site structure shown in atom colored sticks (carbons colour coded as in Fig 2a). Two orientations are displayed as in Fig 2. The omit map for the DMAP substrate is shown as green mesh, contoured at 4 sigma. b) Detailed view of the UbiX^Y169F N5-C1’ adduct species obtained through flash-cooling following reduction. The omit map for the N5-C1’ adduct is shown as green mesh, contoured at 4 sigma.

Extended Data Figure 9

a) DFT models of species II and IVa (as defined in Fig 4). Conversion from II to IVa is achieved by ~180° rotation about C1’-C2’ (blue arrow) and the N5-H and methanol species (red) are only found in species IVa models. b) Overlay of the species II DFT model (green carbons) with the crystal coordinates of species II and Ser15 (teal carbons). c) Three DFT models of IVa were examined and two orthogonal projections are shown overlaid with the crystal coordinates (teal carbons): (Vi, yellow carbons) with a methanol analogue of Ser15 (a, in red) with the C-N5 distance fixed to the crystallographic distance of 4.0 Å; (Vii, magenta carbons) with N5 protonated (no methanol), and (Viii, light pink carbons) with N5 deprotonated and no methanol. DFT model of species V and VI are shown in d) and e), respectively and are overlaid in f) (V green carbons, VI magenta carbons). g). Overlay of the species VI DFT model (magenta carbons) with the crystal coordinates (teal carbons). Models were geometry optimised in the gas phase using the B3LYP/6-311++G(d,p) level of theory. Harmonic vibrational frequencies calculated using normal mode analysis were used to confirm that optimised geometries of all species were in local or global minima. In the case of species Vi, ‘ModRedundant’ optimisation was performed to fix the C-N5 distance and one imaginary frequency of 67.60 cm^-1 was observed. Cartesian coordinates of the optimised structures are given in Supplementary Information.

Fig 1

P. aeruginosa UbiX solutions studies. a) Schematic overview of the proposed UbiX reaction. The N5-C6 prenylated FMNH₂ product (prFMN^reduced) undergoes (likely non-physiological) oxidation to a radical species (prFMN^radical) in presence of oxygen (see panel d). In presence of apo-UbiD or apo-Fdc1, we propose the UbiX product is oxidized to the UbiD/Fdc1 prFMN^iminium cofactor (see panel e). b) Titration of oxidized FMN-UbiX with dimethylallylmonophosphate. Grey lines represent individual scans at increasing [DMAP] concentrations with the black line representing the final spectrum obtained at saturation. An binding curve is derived from the global absorbance change in the 310-450nm range. c) UV-visible spectra of UbiX:FMN obtained during redox cycling in presence of DMAP d) EPR spectrum of the UbiX:prFMN^radical complex e) Reconstitution of A. niger apo-Fdc1 decarboxylase activity by incubation with WT and variant UbiX enzymes in presence of DMAP and FMNH₂ followed by oxygen exposure. No activity can be observed under anaerobic conditions. f) Singular value decomposition of rapid-scan stopped-flow spectrophotometric data following mixing of WT UbiX:FMNH₂ with DMAP. The spectral species identified can tentatively be assigned to 1: ternary UbiX:FMNH₂:DMAP complex, 2: an intermediate covalent adduct formed between FMNH₂ and DMAP and 3: the UbiX:prFMN^reduced product.

Fig 2

Crystal structures of P. aeruginosa UbiX. a) Overview of the dodecameric UbiX structure, with 3 individual monomers (colored in cyan, blue and teal respectively) contributing to a single active site shown in cartoon representation while the remainder (in grey) is shown in surface representation. The bound FMN and DMAP substrates are shown in atom colored spheres. b) Detailed view of the oxidised UbiX:FMN:DMAP complex in two orientations related by 90 degree rotation along the horizontal axis. Individual amino acids contributing to active site structure shown in atom colored sticks (carbons colour coded as in Fig 2a). Residues positioned in front of the FMN:DMAP substrates are shown as thin lines for clarity. The omit map for the DMAP substrate is shown as green mesh, contoured at 4 sigma. Hydrogen bonding networks established with the phosphate moiety are shown by dotted lines. c) Detailed view of the N5-C1’ alkyl adduct species (in two orientations related by a 90 degree rotation along the horizontal axis) obtained through rapid flash-cooling following reduction. The omit map for the N5-C1’ adduct is shown as a green mesh, contoured at 4 sigma. d) Detailed view of the UbiX:product complex in two orientations related by a 90 degree rotation along the horizontal axis. The omit map for the product is shown as green mesh, contoured at 3.5 sigma.

Fig 3

Crystal structures of P. aeruginosa UbiX^E49Q a) Detailed view of the UbiX^E49Q:FMN:DMAP complex with individual amino acids contributing to active site structure shown in atom colored sticks (carbons colour coded as in Fig 2a). Two orientations are displayed as in Fig 2. The omit map for the DMAP substrate is shown as green mesh, contoured at 4 sigma. b) Detailed view of the UbiX^E49Q:FMNH₂:DMAP complex obtained through rapid flash-cooling following reduction. The omit map for the DMAP substrate is shown as green mesh, contoured at 3 sigma. c) Detailed view of the UbiX^E49Q N5-C1’ alkyl adduct species obtained through flash-cooling following reduction. The omit map for the N5-C1’ adduct is shown as green mesh, contoured at 4 sigma.

Fig 4

Schematic representation of the proposed UbiX mechanism. DMAP derived atoms are shown in red. Roman numerals indicate the various intermediate species proposed (see main text). References to individual figures below roman numerals refer to corresponding crystal structures are obtained for the WT or UbiX^E49Q mutant.