The role of UbiX in Escherichia coli coenzyme Q biosynthesis

Abstract

The reversible redox chemistry of coenzyme Q serves a crucial function in respiratory electron transport. Biosynthesis of Q in Escherichia coli depends on the ubi genes. However, very little is known about UbiX, an enzyme thought to be involved in the decarboxylation step in Q biosynthesis in E. coli and Salmonella enterica. Here we characterize an E. coli ubiX gene deletion strain, LL1, to further elucidate E. coli ubiX function in Q biosynthesis. LLI produces very low levels of Q, grows slowly on succinate as the sole carbon source, accumulates 4-hydroxy-3-octaprenyl-benzoate, and has reduced UbiG O-methyltransferase activity. Expression of either E. coli ubiX or the Saccharomyces cerevisiae ortholog PAD1, rescues the deficient phenotypes of LL1, identifying PAD1 as an ortholog of ubiX. Our results suggest that both UbiX and UbiD are required for the decarboxylation of 4-hydroxy-3-octaprenyl-benzoate in E. coli coenzyme Q biosynthesis, especially during logarithmic growth.

Figure 1. Coenzyme Q biosynthetic pathway of Escherichia coli and Saccharomyces cerevisiae

Polyprenyl diphosphate synthases, Coq1p (S. cerevisiae) and IspB (E. coli), catalyze the formation of the isoprenoid tail. The tail lengths among different organisms vary and are specified by the specific polyprenyl diphosphate synthase enzyme present. For example, S. cerevisiae synthesize Q₆, bearing six isoprene units, and E. coli produce Q₈. In S. cerevisiae, the isoprene tail is attached to 4-hydroxybenzoic acid (4-HB) by Coq2p to form 3-hexaprenyl-4-hydroxybenzoate (HP₆B); in E. coli, UbiA catalyzes this reaction to form 4-hydroxy-3-octaprenyl-benzoate (HP₈B). At this point the pathways are proposed to diverge between prokaryotes and eukaryotes. E. coli genes products are identified as Ubi (and include IspB) and S. cerevisiae gene products are identified as COQ.

Figure 2. Expression of yeast PAD1 or E. coli ubiX_K-12 rescues LL1 ubiX null growth on succinate minimal media

Serial 10-fold dilutions (starting A₆₀₀ = 0.1) of the designated bacterial strains were plated on Luria Broth (LB) and succinate minimal media at 30 °C incubated for one day. HW272 (wild-type, parental strain of GD1), and GD1 (ubiG::Kan) were included to provide comparison for growth proficiency and deficiency, respectively. Doubling times were determined in succinate minimal salts liquid media and are presented as (average minutes ± SD) for each of the following strains: MC4100 (wild-type, parental strain of LL1; 70.9 ± 0.2), MC4100/pCH (empty vector; 76.0 ± 9.2), LL1 (ubiX::Kan; 206 ± 12), LL1/pCH (empty vector; 141 ± 22), LL1/pUbiX (plasmid expressing ubiX; 85.3 ± 1.1), and LL1/pYDR538W (plasmid expressing S. cerevisiae PAD1; 91.8 ± 2.7).

Figure 3. Q₈ content in LL1 ubiX null mutant is restored by expression of either E. coli ubiX or S. cerevisiae PAD1

Strains were grown to logarithmic growth (OD₆₀₀ ≈ 0.5) in Luria-Bertani media. Growth media for LL1 were supplemented with kanamycin (50 μg/ml) or ampicillin (100 μg/ml). LL1 strain harboring plasmids expressing the designated genes: pUbiX, ubiX; and pYDR538W, PAD1. The content of Q₈ for each strain was determined by high performance liquid chromatography as described in [11]. Error bars represent standard deviation from three separate injections of the identical sample.

Figure 4. Disruption of the ubiX gene in E. coli leads to an accumulation of radioactive lipid co-eluting with 4-hydroxy-3-octaprenylbenzoate (HP₈B)

Lipid extracts from E. coli strains labeled with p-[U-¹⁴C]hydroxybenzoic acid were separated by normal-phase HPLC. Absorbance was monitored at 274 nm (to detect Q₈) and 250 nm (to detect HP₈B); only one wavelength is depicted and is designated for each of the panels on the left side. The ¹⁴C radioactivity (dpm) present in each fraction is depicted in (B, D, F, H, and J). Unless noted otherwise, the amount of lipid extract injected was 15μl of 500 μl total; chromatograms shown in panels (I and J) represent 15 μl of 50 μl total lipid extract. The paired chromatograms of absorbance and radioactivity (amount of dpm injected is indicated for each panel) correspond to separated lipid extracts from E. coli strains, MC4100 (A and B [6.67 × 10³ dpm]); AN66, ubiD mutant (C and D [2.87 × 10⁴ dpm]); LL1, ubiX mutant with pCH empty vector (E and F [1.87 × 10³ dpm]); LL1 harboring ubiX on a high copy plasmid (G and H [5.02 × 10³ dpm]); and LL1 harboring yeast PAD1 (I and J [4.42 × 10³ dpm]). There was a 0.4 min time delay between the UV and in-line radiochromatography detector.

Figure 5. LC-APCI-MS/MS mass spectra of radiolabeled HP₈B intermediate

The products of the compound designated as 4-hydroxy-3-octaprenyl-benzoate (HP₈B) (see Figure 6) were produced by MS/MS analyses of m/z 683.6. Radiolabeled extracts of AN66 (A) and LL1 (B) were separated by RP-HPLC and MS/MS analysis revealed a transition 683.6 → 150/151 characteristic of the tropylium product ion for HP₈B. For details of the analysis, see “Experimental Procedures.” Treatment with the methylating agent trimethylsilane-diazomethane shifted the tropylium-like ion from 150/151 m/z to 164/165 m/z (Panel insets).

Figure 6. O-methyltransferase activity is decreased in ubiX null in log phase

A) Permeabilized E. coli cells grown to log phase in LB (OD₆₀₀ ≈ 0.5) were prepared from MC4100 (wild-type, parental strain of LL1), LL1 (ubiX::Kan), LL1/pUbiX (plasmid expressing ubiX), and LL1/pYDR538W (plasmid expressing S. cerevisiae PAD1), and incubated with 50 μM of farnesylated DMeQ₃ and 1 mM NADH for 10 min. S-adenosyl-[methyl-³H]-L-methionine (6.9 μM) was added to the reaction mixture. Following incubation for 45 min, lipids were extracted and separated by reverse-phase HPLC. Radioactivity present in fractions 9-11 is expressed as pmol of ³H-CH₃/hr/mg wet weight and eluted with the ³H-product standard. Error bars represent standard deviation from four independent experiments for MC4100 and LL1 and at least 2 independent experiments for LL1/pUbiX and LL1/pYDR538W. An asterisk (*) indicates statistical significance (p = 0.012 by Student's two-tailed t test) when comparing the O-methyltransferase activity of MC4100 and LL1. B) In vitro O-methyltranferase assays were carried out as described in panel A, except MC4100 and LL1 were grown to stationary phase (overnight growth in LB). Error bars represent standard deviation from two separate injections of the identical sample.

Figure 7. Functional roles of UbiD, UbiX, UbiG and 2-octaprenylphenol in Q₈ biosynthesis

During logarithmic growth, both UbiD and UbiX are required to catalyze the decarboxylation of 4-hydroxy-3-octaprenyl-benzoate (HP₈B) and produce 2-octaprenylphenol. Mutations in either UbiD (D) or UbiX (X) result in log phase cells that accumulate HP₈B, and produce only low levels of Q₈. Therefore, the data suggest that both UbiD and UbiX polypeptides are necessary to produce Q₈ in E. coli during log phase growth. For a detailed explanation of the model refer to discussion text. D: UbiD; X: UbiX.