Genetic evidence for an interaction of the UbiG O-methyltransferase with UbiX in Escherichia coli coenzyme Q biosynthesis

Abstract

IS16 is a thiol-sensitive, Q-deficient mutant strain of Escherichia coli. Here, we show that IS16 harbors a mutation in the ubiG gene encoding a methyltransferase required for two O-methylation steps of Q biosynthesis. Complementation of IS16 with either ubiG or ubiX(K-12) reverses this phenotype, suggesting that UbiX may interact with UbiG.

FIG. 1.

Alignment of E. coli UbiG, S. cerevisiae Coq3, human Coq3, and rat COMT amino acid sequences across methyltransferase motifs I, post-I, II, and III. The box designates the L132Q ubiG mutation in the IS16 mutant strain. This amino acid substitution is adjacent to methyltransferase motif II. Sequence analysis revealed that ubiG genes from THU and IS16 share five nucleotide variances in comparison to the ubiG gene from K-12. These variances include T23A, resulting in V8E, and four silent changes, G90T, C109T, A294G, and C321T. Secondary structural elements (β1, β2, β4, and β5) and important active site residues involved in the binding of ligands are indicated for the rat soluble COMT. a, AdoMet; m, magnesium; s, substrate (27).

FIG. 2.

Succinate growth and Q₈ levels in E. coli strains. (A) Serial 10-fold dilutions (starting optical density at 600 nm, 0.2) of the indicated strains were plated on LB and succinate minimal medium plates supplemented with thymine, histidine, and uracil and incubated at 37°C for 1 day. (B) Serial 10-fold dilutions (starting optical density at 600 nm, 0.2) of the indicated strains were plated on LB-AMP and succinate minimal medium plates supplemented with thymine, histidine, and uracil and incubated at 37°C for 1 day. E. coli strains harbored plasmids expressing the designated genes as follows: pAHG, UbiG_K-12; pUbiG_THU, UbiG_THU; pHZ1, UbiX_THU; and pPZ2, UbiX_K-12. (C) Quinones were extracted and separated by reverse-phase HPLC, and Q₈ content was quantified. A standard for Q₈ was prepared from E. coli lipid extracts and verified by mass spectrophotometric methods. External standard curves were created for Q₈ (600, 300, 75, and 30 pmol) and Q₁₀ (300, 150, and 37.5 pmol). The amounts of Q₈ were corrected by recovery of the Q₁₀ internal standard.

FIG. 3.

O methylation of early Q intermediates is defective in IS16 and is restored by expression of E. coli UbiX_K-12. Permeabilized E. coli cells were prepared from HW272 (wild-type, parental strain of GD1), GD1 (ubiG disruption mutant), THU (parental strain of IS16), IS16 (UbiG-L132Q), and IS16:pPZ2 (1S16 harboring UbiX_K-12 on a plasmid) and incubated with farnesylated analogs of 1 mM 3,4-dihydroxy-5-farnesylbenzoic acid (open bars), 250 μM 5-farnesyl-2-hydroxyphenol (closed bars) for 10 min. S-Adenosyl-[methyl-³H]l-methionine (6.9 μM, 81.5 Ci/mmol; PerkinElmer Life Sciences) was added to the reaction mixture. Following incubation at 37°C for 45 min, lipids were extracted and separated by reverse-phase HPLC (BetaBasic C18 column, 5 μM, 4.6 by 250 mm; Thermo Electron Corporation). Radioactivity present in fractions 6 and 7 (open bars) or fractions 10 to 12 (closed bars) is expressed in pmol of CH₃ groups/hr/mg wet weight and eluted with the ³H-labeled product standard. Error bars represent standard deviations obtained from two O-methyltransferase assays of the same sample, and data shown represent two independent experiments.

FIG. 4.

The final O-methylation reaction is defective in the IS16 mutant and is restored by expression of either UbiG or UbiX_K-12. In vitro O-methyltransferase assays were carried out as described for Fig. 3, except that 50 μM 2-farnesyl-5-hydroxy-6-methoxy-3-methyl-1,4 benzoquinone was used as a substrate, and 1 mM NADH was included in all incubations to allow formation of the hydroquinone. At the end of the reaction incubation, 25 μl of freshly prepared 1% ammonium cerium (IV) nitrate was added to the reaction mixture prior to lipid extraction (to oxidize hydroquinone products). The elution positions of the methylated products (fractions 11 and 12) were similar to that of the ³H-labeled product standard and are expressed in pmol of CH₃ groups/hr/mg wet weight. Error bars represent standard deviations obtained from two O-methyltransferase assays of the same sample, and data shown represent two independent experiments. E. coli strains harbored plasmids expressing the designated genes as follows: pAHG, UbiG_K-12; pUbiG_THU, UbiG_THU; pHZ1, UbiX_THU; and pPZ2, UbiX_K-12.

FIG. 5.

Steady-state levels of UbiG and cytochrome o oxidase in E. coli strains. Purified E. coli protein His₆-UbiG (24) was used to generate antisera in rabbits (Cocalico Biologicals, Inc.). Steady-state levels of UbiG were analyzed for E. coli HW272 (wild-type, parental strain of GD1), GD1 (ubiG disruption mutant), THU (parental strain of IS16), IS16 (UbiG-L132Q), and IS16:pPZ2 (IS16 complemented with UbiX_K-12) cells. Cytochrome o oxidase was used as a loading control.

FIG. 6.

Genetic evidence for the interaction of UbiX and UbiG. IS16 harbors a mutation in UbiG (L132Q) that catalyzes the O-methylation step in E. coli coenzyme Q biosynthesis. Lack of growth on succinate, Q deficiency, thiol hypersensitivity (29), and inactive O-methyltransferase activity in IS16 are restored with wild-type ubiX_K-12, suggesting that UbiX may be interacting with UbiG (L132Q). X, UbiX; G, UbiG.