(Received for publication, November 21, 1996, and in revised form, January 17, 1997)
From the Department of Chemistry and Biochemistry and the Molecular
Biology Institute, UCLA, Los Angeles, California 90095 and the
Department of Biological Sciences, Columbia University,
New York, New York 10027
Ubiquinone (coenzyme Q or Q) is a lipophilic metabolite that functions in the electron transport chain in the plasma membrane of prokaryotes and in the inner mitochondrial membrane of eukaryotes. Q-deficient mutants of Saccharomyces cerevisiae fall into eight complementation groups (coq1-coq8). Yeast mutants from the coq5 complementation group lack Q and as a result are respiration-defective and fail to grow on nonfermentable carbon sources. A nuclear gene, designated COQ5 was isolated from a yeast genomic library based on its ability to restore growth of a representative coq5 mutant on media containing glycerol as the sole carbon source. The DNA segment responsible for the complementation contained an open reading frame (GenBankTM accession number Z49210[GenBank]) with 44% sequence identity over 262 amino acids to UbiE, which is required for a C-methyltransferase step in the Q and menaquinone biosynthetic pathways in Escherichia coli. Both the ubiE and COQ5 coding sequences contain sequence motifs common to a wide variety of S-adenosyl-L-methionine-dependent methyltransferases. A gene fusion expressing a biotinylated form of Coq5p retains function, as assayed by the complementation of the coq5 mutant. This Coq5-biotinylated fusion protein is located in mitochondria.
The synthesis of two farnesylated analogs of intermediates in the ubiquinone biosynthetic pathway is reported. These reagents have been used to develop in vitro C-methylation assays with isolated yeast mitochondria. These studies show that Coq5p is required for the C-methyltransferase step that converts 2-methoxy-6-polyprenyl-1,4-benzoquinone to 2-methoxy-5-methyl-6-polyprenyl-1,4-benzoquinone.
Ubiquinone (coenzyme Q or Q)1 is a
lipid that consists of a quinone head group and a polyprenyl tail that
varies in length depending on the organism. The primary function of Q
is to transport electrons from Complex I or II to the cytochrome
bc1 complex in the inner mitochondrial membrane
of eukaryotes and the plasma membrane of prokaryotes (1). This cycle is
carried out by a series of reductions and oxidations of the head group
of Q. This same redox chemistry also allows reduced Q (QH2)
to scavenge electrons and function as a lipid-soluble antioxidant. In
this latter capacity, QH2 may scavenge lipid peroxyl
radicals directly in an analogous manner to vitamin E (2-4), or it may
help regenerate -tocopherol (5, 6). Recent evidence suggests that
QH2 plays an important role in vivo in
protecting cells from autoxidation products of polyunsaturated fatty
acids (7). Higher levels of QH2 in low density lipoproteins
usually correspond to an increased resistance to lipid peroxidation
(8-11). It has been suggested that the level of antioxidants, such as
QH2, in low density lipoproteins may slow the development
of atherosclerosis, since oxidatively modified low density lipoproteins
are thought to play a role in the initiation of the disease (12, 13).
This action of QH2 as an antioxidant may also impact other
age-related degenerative diseases and the aging process itself (14,
15).
Q is synthesized from p-hydroxybenzoic acid and polyisoprenediphosphate in both prokaryotes and eukaryotes (16). The proposed pathway for the biosynthesis of Q was elucidated from the characterization of Q intermediates that accumulated in Q-deficient strains of both Saccharomyces cerevisiae and Escherichia coli (16, 17). It was shown that the pathways in these organisms diverge after formation of 3-polyprenyl-4-hydroxybenzoate. After three additional steps, the Q biosynthetic pathways in E. coli and S. cerevisiae are thought to converge. The ubi mutants of E. coli are grouped into eight complementation groups (ubiA-ubiH) and fail to grow on media containing succinate as the sole carbon source (17, 18). The Q-deficient strains of S. cerevisiae have also been grouped into eight complementation groups (coq1-coq8) and are nonrespiring and therefore fail to grow on nonfermentable carbon sources (19). The Q deficiency in the coq complementation groups was based on the observation that in vitro assays of cytochrome c reductase activity could be returned to almost wild-type levels by the addition of Q (20). Yeast coq mutant strains (representative mutants from coq3-coq8 groups) when grown in the presence of p-[U-14C]hydroxybenzoic acid, a biosynthetic precursor of Q, fail to produce Q and accumulate an early intermediate that corresponds to 3-hexaprenyl-4-hydroxybenzoic acid (21). The presence of this intermediate is not necessarily diagnostic of the affected biosynthetic step, since it also accumulates in wild-type yeast (21, 22). This phenomenon is not observed in the E. coli Q-deficient ubi mutants, which tend to accumulate large amounts of each distinct Q intermediate (17).
The current study employs the strategy of comparing both sequence and
function of the yeast COQ and E. coli ubi gene
products to study the C-methylation step in yeast Q
biosynthesis (depicted in Fig. 1). This C-methyltransferase
step was identified as being potentially defective in E. coli
ubiE mutants, which accumulate the intermediate DDMQ (Fig. 1; Ref.
23). Searches of the literature have shown very few examples of
C-methyltransferases, with most of the examples being in
DNA/RNA and in steroid, corrin, and porphyrin biosynthesis (24).
Recently, the E. coli ubiE gene has been identified and
shown to harbor three sequence motifs common to a wide variety of
S-adenosyl-L-methionine-dependent
methyltransferase enzymes (25). The presence of an intact
ubiE gene was required for production of Q in
vivo, and disruption of the ubiE gene in a wild-type
parental strain produced a mutant with growth defects on succinate and
in both Q and menaquinone synthesis (25). The present study reports the
isolation of a ubiE homolog from yeast, the COQ5
gene, and provides evidence that it encodes a
C-methyltransferase. To study the
C-methylation step shown in Fig. 1,
farnesylated analogs of the natural substrate and product were
synthesized and used to develop an in vitro assay. The data
presented indicate that the yeast Coq5 polypeptide is located in yeast
mitochondria and is required for C-methylation in the
synthesis of Q.
The genotypes and sources of the mutant and wild-type yeast strains used in this study are shown in Table I. Media for growth of yeast were prepared as described (29) and included YPD (1% yeast extract, 2% peptone, 2% dextrose), YPG (1% yeast extract, 2% peptone, 3% glycerol), YPGal (1% yeast extract, 2% peptone, 2% galactose), and YEPG (1% yeast extract, 2% peptone, 2% ethanol, 3% glycerol). The SD medium (0.18% yeast nitrogen base without amino acids, 2% dextrose, 0.14% NaH2PO4, 0.5% (NH4)2SO4) was modified from the literature as described. Amounts of adenine, uracil, tryptophan, histidine HCl, and methionine were increased by a factor of 4; amounts of arginine HCl, isoleucine, and lysine HCl were doubled; phenylalanine and leucine were increased by 120%; tyrosine was increased by 130%; 80 mg/liter cysteine was added. SD-Leu and SD-Ura were SD media without leucine or uracil, respectively. 2% agar was added for solid media. Yeast were grown at 30 °C. The E. coli strain AN70 (ubiE401) harboring a mutation in the ubiE gene (23) was grown as described previously (25).
|
C83/LH1 was transformed by the procedure of Beggs (30) with 5 µg of a yeast genomic library. The library was constructed by ligation to the BamHI site of YEp13 of partial Sau3AI fragments (7-10 kb) of nuclear DNA isolated from the respiration-competent S. cerevisiae strain D273-10B. Two respiration-competent and leucine prototrophic clones were obtained from the transformation. Both phenotypes co-segregated, indicating their dependence on the presence of the autonomously replicating plasmid. The plasmid pG17/T1, isolated from one of the transformants, was used to characterize the complementing gene.
In a similar manner, CH83-B3 was transformed with the multiple-copy
expression library prepared from yeast DNA in the vector YEp24 (31)
containing the URA3 gene as a selectable marker. Transformants were selected by plating onto SD-Ura medium and replica-plated after 2 days onto YPG medium to test for respiratory growth. One respiration-competent and uracil-prototrophic colony was
obtained from the transformation, and the co-segregation of these
phenotypes indicated the presence of a plasmid gene. Yeast plasmid DNA
was recovered from the transformant and amplified in E. coli
DH5 (Life Technologies, Inc.). Restriction enzyme mapping and
Southern hybridization analysis of the recovered plasmid, pRB01, showed
it to contain a segment of DNA that overlapped with the insert present
in pG17/T1.
pG17/ST1 was obtained by religation of pG17/T1 after removal of the 2-kb SphI fragment. The 2-kb SphI fragment containing one-half of the pG17/T1 insert and 200 base pairs of YEp13 sequence were subcloned into the vector YEp351 (32) to create pG17/ST2. Partial DNA sequence analysis of the 0.8-kb EcoRI fragment derived from pG17/ST1 showed the insert DNA to contain a segment of the chromosome XIII sequence reported by Skelton et al.2 Based on the sequence of YEp13 and the reported open reading frame (accession number Z49210[GenBank]), the removal of the 2-kb SphI fragment leads to the loss of the 28 amino-terminal residues and their substitution by the following sequence: MTQSAAGTCPTSCMIKKTVISAATIVMPRAHRKELTGLKALKGIGRRSPLCDSCIRKQPSSRLRPLSTAAARNG.
The disrupted coq5 allele was constructed in pG17/T1 by
substituting the 100-base pair BamHI fragment internal to
the gene with HIS3 on a 1.7-kb BamHI fragment
(34). The resultant plasmid, pG17/ST3, was used to obtain a linear
3.5-kb HincII fragment with the disrupted gene. W303-1A and
W303-1B were each transformed with 2 µg of DNA by the procedure of
Schiestl et al. (35). Most of the histidine-independent
clones issued from the transformations were respiration-defective and
were complemented by 0 but not by the coq5
testers. Nuclear DNAs from two independent transformants, one with an
"a" and the other with an
mating type were digested with
EcoRI and separated on 1% agarose. Southern analysis of
these independent transformants confirmed the presence of the
coq5::HIS3 allele in their chromosomal DNAs. The
probe, an 800-base pair EcoRI fragment (Fig.
2) recognizes a 800-base pair fragment in the genomic
DNA of the parental strain. The genomic DNA of the mutant strain,
aW303
COQ5, has a larger hybridizing species at approximately 2.5 kb,
as expected for the disrupted allele.
Construction of Biotinylated Coq5p
The gene for
biotinylated Coq5p was constructed by in-frame fusion of a polymerase
chain reaction-amplified fragment containing the 5-flanking and coding
sequence to a bacterial sequence coding for the biotinylation site of
transcarboxylase (36). The amplification was carried out with pG17/T1
as template, a forward primer (encoding the SacI site)
starting at nucleotide position
373, and a reverse primer starting at
+907, in which the termination codon of COQ5 was destroyed
and replaced with a BglII site. The polymerase chain reaction product was digested with a combination of
SacI and BglII and ligated to the
BamHI and SacI sites in the yeast/E.
coli shuttle vector YEp352-Bio6 (33, 58). The fusion gene was
introduced into W303
COQ5 either on an autonomously replicating
plasmid (pG17/ST5) or by integration of a single copy at the URA3
locus (pG17/ST6).
The wild-type strain
W303-1B and the transformants W303COQ5/ST5 and aW303
COQ5/ST5
were grown in YPGal (2% galactose, 1% yeast extract, 2% peptone) to
early stationary phase. The cells were converted to spheroplasts by
digestion with Zymolyase 20,000. Spheroplasts were lysed in 0.6 M sorbitol and debris, and unlysed cells were removed by
centrifugation at 2,000 × g for 10 min. The
mitochondria were sedimented at 12,000 × g for 15 min.
Total mitochondrial and postmitochondrial supernatant proteins (20 µg) were loaded in each lane and separated in a 12% polyacrylamide gel prepared according to Laemmli (37). The proteins were transferred electrophoretically to nitrocellulose, and the Western blot was reacted
first with avidin coupled to peroxidase. The blot was then stained with
4-chloro-1-naphthol in the presence of hydrogen peroxide. This
procedure stains only proteins containing biotin.
All reagents for organic synthesis were purchased from Aldrich and used as received unless otherwise stated. Dichloromethane, hexamethylphosphoric triamide, and triethylamine were distilled from calcium hydride. Diethylether was distilled from sodium-benzophenone ketyl. Unless specified as dry, the solvents were of unpurified reagent grade. All air- or water-sensitive reactions were carried out under positive pressure of argon in oven-dried glassware. Reactions were followed by TLC using Whatman precoated plates of silica gel 60 with fluorescent indicator (0.25 mm). Reactions forming quinones were followed by leucomethylene blue stain. Flash chromatography was performed on Davisil Grade 643 silica gel (230-400 mesh). NMR spectra were measured on a Bruker AM360, ARX400, or ARX500 spectrometer and were recorded in ppm using the solvent signal as an internal standard. Mass spectra and high resolution mass spectra were recorded on a VG Autospec and are reported in units of mass to charge (m/z). High resolution mass spectra were recorded with an EI source.
2,5-Diacetoxy-3-methoxybromobenzene (Fig. 6, 2)Vanillin (1) (2.00 g, 13.1 mmol) was
dissolved in acetic acid (20 ml). Bromine (15.8 mmol, 1.0 M
solution in CCl4) was added slowly, and the reaction
mixture was allowed to stir at room temperature overnight (12 h). Water
was then added (20 ml), and the CCl4 was removed in
vacuo. The aqueous layer was adjusted to pH 5 with 1 M
NaOH and extracted with ethyl acetate (2 × 20 ml). The combined
organic extracts were washed with water (2 × 20 ml), 1.0 M NaHCO3 (20 ml), and brine (20 ml), dried over MgSO4, and filtered. After concentration, a combined mass
of 2.61 g (86% yield) of orange crystalline
3-bromo-4-hydroxy-5-methoxybenzaldehyde was obtained. Mp:
157-159 °C. 1H NMR (CDCl3, 360 MHz) :
3.98 (s, 3H); 6.54 (br s, 1H); 7.36 (d, 1H, J = 1.7 Hz); 7.64 (d, 1H, J = 1.7 Hz); 9.78 (s, 1H).
13C NMR (CDCl3, 90 MHz)
: 56.59; 107.95;
108.13; 129.98; 130.09; 148.85; 149.32; 189.70. IR (thin film on NaCl)
(cm
1): 3281; 1674; 1589; 1424; 1354; 1291; 1157; 1046;
681. HRMS calculated (Calcd) for
[C8H7BrO3]+:
229.9579. Found: 229.9582.
The reaction vessel for the next step was soaked prior to reaction for
24 h in 0.1 M NaOH/H2O2
solution and rinsed with H2O (38).
3-Bromo-4-hydroxy-5-methoxybenzaldehyde (1.00 g, 4.33 mmol) was
dissolved in H2O (20 ml) and treated with NaOH (381 mg,
9.53 mmol). Hydrogen peroxide (30% solution in H2O; 1.08 ml, 9.53 mmol) was dissolved in H2O (20 ml) and added
dropwise to the reaction mixture. The reaction was stirred at room
temperature for 90 min. The reaction mixture was then acidified to pH 3 with 10% HCl and extracted with ethyl acetate (2 × 40 ml). The
combined extracts were washed with brine (40 ml) and dried over
MgSO4. Flash chromatography (RF 0.3, 7:3 hexanes/ethyl acetate) was performed to give 557 mg (59% yield) of
tan solid 2-bromo-4-hydroxy-6-methoxy phenol. 1H NMR
(Me2SO-d6, 360 MHz) : 3.69 (s,
3H); 6.43 (d, 1H, J = 2.4 Hz); 6.46 (d, 1H,
J = 2.3 Hz); 8.57 (br s, 1H); 9.13 (br s, 1H). 13C NMR (Me2SO-d6, 90 MHz)
: 55.90; 99.96; 109.48; 109.62; 136.38; 149.20; 150.64. IR
(thin film on NaCl) (cm
1): 3258; 1615; 1470; 1429; 1294;
1190; 1134; 1035; 828. HRMS Calcd for
[C7H7BrO3]+:
217.9579. Found: 217.9577.
2-Bromo-4-hydroxy-6-methoxy phenol (250 mg, 1.15 mmol) was suspended in
CH2Cl2 (2.5 ml). Triethylamine (0.56 ml, 4.12 mmol) and acetic anhydride (0.33 ml, 3.44 mmol) were then added,
followed by 4-(dimethylamino)pyridine (~15 mg). The reaction was
allowed to proceed for 7 h at room temperature. The reaction was
quenched with NH4Cl, and the CH2Cl2
was removed in vacuo. The residue was extracted with ethyl
acetate (2 × 5 ml), and the combined extracts were washed with
brine (5 ml), dried over MgSO4, filtered, and concentrated.
Flash chromatography (R factor 0.3, 8:2 hexanes/ethyl acetate) was performed to obtain 330 mg (95% yield) colorless oil
2 in Fig. 6. 1H NMR (CDCl3, 360 MHz)
: 2.25 (s, 3H); 2.22 (s, 3H); 3.77 (s, 3H); 6.68 (d, 1H,
J = 1.8 Hz); 6.95 (d, 1H, J = 1.8 Hz).
13C NMR (CDCl3, 90 MHz)
: 20.21; 20.82;
56.16; 105.65; 116.73; 117.19; 135.56; 148.66; 152.48; 167.65; 168.73. IR (thin film on NaCl) (cm
1): 3020; 1771; 1601; 1482;
1412; 1369; 1204; 1175; 1134; 1044; 1015. HRMS Calcd for
[C11H11BrO5]+:
301.9790. Found: 301.9792.
Tetrakis(triphenylphosphine)palladium
(0) (Pd(PPh3)4; 23 mg, 0.020 mmol) was
dissolved in hexamethylphosphoric triamide (HMPA; 1.0 ml), in a Schlenk
tube under argon in a glove box. Compound 2 (305 mg, 1.00 mmol) was dissolved in HMPA (1.0 ml), and the resulting solution was
added via syringe to the solution containing Pd(PPh3)4 (39). A solution of farnesyl
tributylstannane (743 mg, 1.5 mmol) (40) dissolved in HMPA (1.5 ml) was
then added to this reaction mixture. The reaction vessel was sealed
under argon, removed from the glove box, and heated at 65 °C in a
sand bath for 48 h. The reaction was then quenched with saturated
NH4Cl (3 ml) and extracted with ether (3 × 5 ml). The
ethereal layers were washed with H2O (5 ml) and brine (5 ml), dried over MgSO4, filtered, and concentrated. Flash
chromatography was performed (RF 0.5, 9:1
hexanes/ethyl acetate) to give 322 mg (75% yield) of pale yellow oil
1,4-diacetoxy-2-farnesyl-6-methoxybenzene. 1H NMR
(CDCl3, 360 MHz) : 1.60 (s, 6H); 1.60 (s, 6H); 2.05 (m, 8H); 2.26 (s, 3H); 2.29 (s, 3H); 3.22 (d, 2H, J = 7.2 Hz); 3.77 (s, 3H); 5.09 (m, 2H); 5.21 (m, 1H); 6.55 (d, 1H,
J = 2.6 Hz); 6.58 (d, 1H, J = 2.6 Hz).
13C NMR (CDCl3, 90 MHz)
: 13.55; 15.93;
16.12; 17.17; 17.62; 20.39; 21.07; 25.61; 26.42; 26.69; 26.79; 27.79;
28.41; 39.58; 39.66; 56.00; 104.04; 113.79; 120.71; 123.97; 124.33;
131.21; 135.09; 135.49; 135.60; 137.32; 148.47; 151.49; 168.63; 169.32. IR (thin film on NaCl) (cm
1): 2921; 1767; 1483; 1466;
1427; 1370; 1210; 1175; 1136; 1020. HRMS Calcd for
[C26H36O5]+:
428.2563. Found: 428.2571.
LiAlH4 (67 mg, 1.75 mmol) was suspended in ether (5 ml) and
cooled to 0 °C. 1,4-Diacetoxy-2-farnesyl-6-methoxybenzene (150 mg,
0.350 mmol) was dissolved in ether (10 ml) and added dropwise to the
suspension. The resulting reaction mixture was stirred for 1 h.
The reaction was quenched with water (5 ml) and then 1 M
NaOH (5 ml), followed by water (5 ml) and allowed to warm to room
temperature. A colorless precipitate formed and was extracted with
ether (2 × 15 ml), and the combined ethereal extracts were washed
with brine (30 ml), dried over MgSO4, filtered, and
concentrated. The expected hydroquinone was oxidized during basic
work-up, and a bright yellow oil (3) was obtained (110 mg,
92% yield). 1H NMR (CDCl3, 360 MHz) : 1.57 (s, 6H); 1.61 (s, 3H); 1.65 (s, 3H); 2.02 (m, 8H); 3.11 (d, 2H,
J = 7.2 Hz); 3.79 (s, 3H); 5.06 (m, 2H); 5.13 (m, 1H);
5.85 (d, 1H, J = 2.4 Hz); 6.42 (d, 1H, J = 2.4 Hz). 13C NMR (CDCl3, 90 MHz)
: 15.96; 16.08; 17.60; 25.63; 26.32; 26.61; 27.07; 39.56 (two
signals); 56.21; 101.01; 117.62; 123.67; 124.27; 131.18; 132.73;
135.33; 140.05; 146.34; 158.80; 182.11; 187.64. IR (thin film on NaCl)
(cm
1): 3451; 2919; 2851; 1682; 1651; 1603; 1454; 1233;
1176; 1045; 911; 773. HRMS Calcd for
[C22H30O3]+:
342.2195. Found: 342.2192. UV
max (nm): 264, 206.
To a
solution of KH2PO4 (4 g, 29.4 mmol) in 400 ml
of water was added freshly prepared potassium nitrosodisulfonate (6 g, 22.4 mmol) (41, 42). To this purple solution, 2-methoxy-5-methylaniline (4) (1.68 g, 12.2 mmol) in 50 ml of acetone was added. The
reaction was stirred at 23 °C for 1.5 h, during which the color
changed to reddish green. The reaction was extracted three times with
100 ml of chloroform. The chloroform was dried over MgSO4,
filtered, and concentrated by rotary evaporation. The resulting solid
was washed five times with cold ether (50-ml aliquots), leaving a
yellowish green solid (5) (1.21 g, 7.95 mmol, 65%):
1H NMR (CDCl3, 500 MHz) : 6.55 (d, 1H,
J = 1.6 Hz), 5.92 (s, 1H), 3.18 (s, 3H), 2.06 (d, 3H,
J = 1.6 Hz); 13C NMR (CDCl3,
125 MHz)
: 187.66, 182.13, 158.71, 146.87, 131.26, 107.56, 56.22, 15.78; EIMS 152.05 [M+], 137.03, 122.04; HRMS Calcd for
[C8H8O3]+:
152.047344. Found: 152.047276.
To a solution of compound 5 (120 mg,
0.8 mmol) in 10 ml of dry 1,4-dioxane was added zinc powder (200 mg, 3 mmol) and 4 drops of concentrated HCl. The HCl was added dropwise
followed by shaking until the solution turned nearly colorless. The
zinc was precipitated by centrifugation, and the supernatant was
transferred to a separate flask. To this flask was added
BF3-OEt2 (0.89 g, 6.3 mmol) followed by
trans,trans-farnesol (0.35 g, 1.6 mmol). The
reaction was stirred at 23 °C for 16 h. The reaction was
quenched by the addition of 20 ml of 1 M
NaHCO3. The reaction was extracted three times with ether,
and the combined organic layers were dried over MgSO4,
filtered and concentrated by rotary evaporation. The remaining oil was
taken up in 15 ml of benzene, and to it was added FeCl3
(400 mg, 2.5 mmol) in 15 ml of water. This reaction was allowed to stir
for 3 h, and then the organic layer was extracted, washed three
times with water, dried over MgSO4, filtered, and concentrated by rotary evaporation. The crude product was purified by
flash chromatography (9:1 hexanes:ethyl acetate) to afford 40 mg of
both demethoxyubiquinone-3 and isodemethoxyubiquinone-3 (14.2% yield
for each). TLC: RF 0.33 (3:1 hexanes:ethyl
acetate); 1H NMR (CDCl3, 400 MHz) : 5.87 (s,
1H), 5.06 (m, 2H), 4.94 (t, 1H, J = 1.2 Hz), 3.78 (s,
3H), 3.21 (d, 2H, J = 7 Hz), 2.04 (s, 3H), 1.93-2.10
(m, 8H), 1.74 (s, 3H), 1.67 (s, 3H), 1.58 (s, 3H), 1.56 (s, 3H);
13C NMR (CDCl3, 100 MHz)
: 187.81, 181.89, 158.34, 141.80, 141.34, 137.69, 135.2, 131.29, 124.33, 123.85, 118.80,
107.00, 56.11, 39.69, 29.24, 26.74, 26.43, 25.71, 25.27, 17.68, 16.34, 16.02, 12.19; EIMS 356.2 [M+], 267.0, 245.1, 220.1, 205.1, 189.2, 167.1, 136.1, 121.1; HRMS Calcd for
[C23H32O3]+:
356.235145. Found: 356.235365.
TLC:
RF 0.31 (9:1 hexanes:ethyl acetate);
1H NMR (CDCl3, 400 MHz) : 6.42 (d, 1H,
J = 1.6 Hz), 5.06 (m, 3H), 4.00 (s, 3H), 3.15 (d, 2H,
J = 7.3 Hz), 2.03 (d, 3H, J = 1.6 Hz),
1.93-2.10 (m, 8H), 1.74 (s, 3H), 1.67 (s, 3H), 1.58 (s, 3H), 1.56 (s,
3H); 13C NMR (CDCl3, 100 MHz)
: 188.05, 184.08, 155.16, 145.65, 137.06, 135.05, 131.92, 131.44, 131.28, 124.32, 123.98, 119.94, 60.97, 39.69, 26.73, 26.46, 25.68, 22.55, 17.67, 16.15, 15.99, 15.82; EIMS 356.2 [M+], 317.0, 267.0, 245.1, 220.1, 205.1, 189.2, 162.0, 136.1; HRMS Calcd for
[C23H32O3]+:
356.235145. Found: 356.234814.
Mitochondria were isolated by the method of
Daum et al. (43) except that Oxalyticase (Enzogenetics) was
substituted for Zymolyase. Mitochondria were either used immediately or
stored for later use at 80 °C after being frozen in liquid
nitrogen. Protein concentration was determined with the BCA method
(Pierce). Each methylation reaction (250 µl) contained 1 mM ZnSO4, 500 µM substrate
(2-methoxy-6-farnesyl-1,4-benzoquinone) in methanol (5 µl total), 100 µl of yeast mitochondria (containing 1-4 mg of total protein), and
0.05 M sodium phosphate, pH 7.0. The concentration of NADH
was 3.0 mM in reactions where it was required. The reaction was initiated by the addition of
S-adenosyl-[methyl-3H]L-methionine
to a final concentration of 20 µM (DuPont NEN, 84.1 Ci/mmol; specific activity adjusted to 560 mCi/mmol with unlabeled
S-adenosyl-L-methionine;
15,200 M
1 cm
1, 256 nm, pH 1; Ref. 44).
Following a 1-h incubation at 30 °C, the reaction was quenched by
the addition of 2 µl of glacial acetic acid. The lipids were
extracted with pentane (500 µl, twice), concentrated, and resuspended
in methanol. The extracts were analyzed on a reverse-phase HPLC column
(Alltech Lichrosorb C-18; 5 µm, 4.6 × 250 mm) with 9:1
methanol:water as a mobile phase at a rate of 1 ml/min (22). Each 1-ml
fraction was analyzed for radioactivity by scintillation counting with
Safety Solve (Research Products International Corp.) as a scintillation
fluor.
E. coli cell-free extracts were prepared as described previously (45). Each reaction was carried out the same way as described for the yeast mitochondrial in vitro assay with the following changes: 1) the DDMQ substrate concentration was reduced to 100 µM; and 2) following a 1-h incubation at 37 °C, lipids were extracted with hexane (0.5 ml, twice), concentrated, and resuspended in methanol.
The coq5
complementation group (G17) is composed of 20 independent isolates that
contain recessive nuclear mutations resulting in the loss of
respiration and Q synthesis (19). To learn more about the affected step
in Q biosynthesis in the coq5 mutant, the COQ5
gene was cloned by transformation of either C83/LH1 or CH83-B3 with
yeast genomic DNA as described under "Materials and Methods." Three
respiration-competent transformants were obtained, one of which was
used to characterize the complementing gene. This transformant
contained a plasmid with a nuclear DNA insert of 3.8 kb. A partial
restriction map is presented in Fig. 2. Subcloning analysis of the
insert of pG17/T1 indicated that pG17/ST2 did not complement C83/LH1,
while pG17/ST1 did complement, but the growth of these transformants on
glycerol was slower than the ones containing pG17/T1. DNA sequence
analysis of part of the 0.8-kb EcoRI fragment identified an
open reading frame encoding a 34.7-kDa hypothetical protein (Fig.
3), located on chromosome XIII as reported by Skelton
et al.2 Based on this sequence, the construction
of the pG17/ST1 clone leads to the loss of 28 amino-terminal residues
from the coding region indicated in Fig. 3 and their substitution with
a 74-residue amino-terminal extension derived from YEp13 DNA (see
"Materials and Methods"). The expression of this modified version
of Coq5p is presumably responsible for the slow growth phenotype
observed when the CH83/LH1 mutant strain is transformed with pG17/ST1
multicopy plasmid. Analysis of the COQ5 open reading frame
identified three methyltransferase sequence motifs (Fig. 3) that are
present in a large family of methyltransferases that use
S-adenosyl-L-methionine as the methyl donor
(24). The amino acid sequence of the Coq5 polypeptide is 44% identical
over 262 amino acids with the E. coli UbiE
polypeptide, which is required for the C-methylation reaction in Q biosynthesis (25). This degree of sequence identity and
the presence of the methyltransferase motifs suggests that the
Coq5 polypeptide is likely to function as a
C-methyltransferase in eukaryotic Q biosynthesis.
In Situ Disruption of the COQ5 Gene
The one-step gene
replacement procedure (34) was used to obtain the coq5 null
mutant strains aW303COQ5 and W303
COQ5, as described under
"Materials and Methods." These yeast strains are both
histidine-prototrophic and respiration-deficient and are complemented
by a
0 strain but not by other G17 mutants. These
results imply a genetic linkage of the coq5::HIS3
null and the coq5-1 mutant alleles. To verify this, C83/LH1
was transformed with a linear fragment of DNA containing the
coq5::HIS3 allele. Two histidine-prototrophic transformants obtained from the transformation were crossed to W303-1A. Diploid cells obtained from the crosses were sporulated on
potassium acetate plates and subjected to tetrad analysis. Meiotic
progeny from 11 complete tetrads derived from each cross were tested
for respiration and histidine dependence. Both phenotypes segregated
2:2 in the 22 tetrads analyzed. In all cases, the respiration-deficient spores were histidine-independent while the respiration-competent spores were histidine auxotrophs, confirming the allelism between the cloned COQ5 gene and the original coq5-1
mutation.
The
intracellular localization of Coq5p was studied in cells expressing the
COQ5 gene fused in-frame with a sequence coding for the
biotinylation signal of a bacterial transcarboxylase. The fusion gene
(COQ5-BIO) was expected to express a hybrid protein consisting of the native sequence of Coq5p, with a carboxyl-terminal extension of 78 residues containing covalently attached biotin. Transformation of the W303COQ5 null mutant with the fusion gene in
the multicopy plasmid pG17/ST5 conferred wild-type growth properties on
the nonfermentable substrate glycerol (Fig. 4). The
fusion gene also complements when inserted into chromosomal DNA in
single copy, although the latter transformants grow more slowly on
glycerol. These results indicate the carboxyl-terminal addition of the
bacterial sequence with covalently attached biotin only partially
compromises the activity of the protein.
The distribution of biotinylated Coq5p was examined in transformants
overexpressing the fusion protein from the multicopy plasmid pG17/ST5.
Total mitochondrial and postmitochondrial proteins of the parental wild
type and two independent transformants (W303COQ5/ST5 and
aW303
COQ5/ST5) were separated by SDS-polyacrylamide gel
electrophoresis and transferred to nitrocellulose. Biotinylated
proteins were detected by probing the Western blot with peroxidase
coupled to avidin. These analyses indicated the presence of an abundant
biotinylated protein in the transformants with an estimated mass of
40-41 kDa (Fig. 5). This protein is not detected in the
postmitochondrial supernatant fraction, suggesting a
mitochondrial localization. The size of the biotinylated Coq5p measured
from its migration is in good agreement with a molecular weight of
42,302 calculated from the amino acid composition. Since the
amino-terminal sequence has the characteristic features of
mitochondrial import signals (46), it is likely that the primary
translation product is proteolytically cleaved during transport into
mitochondria, thus accounting for the slight difference in size.
C-Methylation Assays
To determine whether the Coq5
polypeptide is required for the C-methylation of DDMQ to
form DMQ, farnesylated analogs of both were synthesized chemically
(Fig. 6). The farnesylated analog of DDMQ (3)
was tested as a substrate in an in vitro methylation assay
(Fig. 7). Mitochondria isolated from the parental wild
type (W303-1B) were incubated with 3 and the methyl donor
S-[methyl-3H]adenosyl-L-methionine,
and the organic extract was analyzed by reverse-phase HPLC. The
radioactive methylated product co-eluted with the corresponding
chemically synthesized methylated product DMQ (Fig. 7A).
In vitro assays performed with mitochondria isolated from
the coq5 null mutant (W303COQ5) produced no detectable
methyltransferase activity over background (Fig. 7B).
Transformation of the coq5 null mutant with the multicopy
plasmid containing the yeast COQ5 gene restored the ability
to form the methylated product and in fact generated a significantly
higher level of activity (0.38 pmol of product ·mg of
protein
1·h
1) as compared with wild type
(0.05 pmol of product·mg of protein
1·h
1
(Fig. 7C). Assays in which either mitochondria or substrate
(3) were omitted showed no methyltransferase activity. NADH
was a necessary component of the assays, since in the absence of NADH the methyltransferase activity was reduced. The addition of NADH to the
in vitro methylation reaction restored activity to normal levels (data not shown). A similar dependence of methylated product formation on the presence of the UbiE polypeptide was observed in
in vitro C-methyltransferase assays with E. coli
cell lysates. Cell-free extracts prepared from the E. coli strain AN70 harboring a mutation in the ubiE gene
fail to generate detectable methylated product, while the same mutant
rescued with a plasmid harboring the ubiE gene
produces a methylated product that co-elutes with the DMQ standard
(data not shown).
This study describes the isolation and characterization of the COQ5 gene. The COQ5 gene was recovered from a yeast genomic library on the basis of its ability to restore growth of a coq5 mutant strain on glycerol, a nonfermentable carbon source. A segment of DNA sequence from the region responsible for complementation was identical to a previously reported S. cerevisiae open reading frame encoding a hypothetical 34.7-kDa protein.2 This open reading frame contained 44% identity over 262 amino acid residues to UbiE, which is required for the C-methylation reactions in both Q and menaquinone biosynthesis in E. coli (23, 25, 47). Both the E. coli UbiE and yeast Coq5 amino acid sequences contain three structural motifs that appear to be conserved in most methyltransferases that use S-adenosyl-L-methionine as a methyl donor (24). Recent crystal structures suggest that these motifs either contact S-adenosyl-L-methionine or are used as structural scaffolding for other residues that contact it (48). Based on the presence of these motifs, and the degree of sequence identity between Coq5p and UbiE, it seems likely that COQ5 and ubiE correspond to the structural genes encoding the C-methyltransferase enzyme in Q biosynthesis in yeast and E. coli, respectively.
The in vitro methyltransferase assays provide further evidence that Coq5p and UbiE catalyze a C-methyltransferase step in Q biosynthesis. These assays employed farnesylated analogs of both DDMQ and DMQ, and the syntheses presented for these two analogs yield large amounts of product from very inexpensive starting materials. The data presented indicate that both Coq5p and UbiE catalyze the step converting DDMQ to DMQ. These studies suggest that the length of the isoprene tail does not play a crucial role in substrate recognition, since normal substrates for this C-methylation contain eight and six isoprene units, in E. coli and S. cerevisiae, respectively. Several other studies also indicate the isoprenoid tail length does not play a crucial role in enzyme recognition (45, 49-51). In addition, a respiration-competent S. cerevisiae strain has been generated that produces only Q8, indicating that a specific tail length of Q is not a crucial aspect of either the biosynthesis of Q or its respiratory function in vivo (52).
The in vitro assays with yeast mitochondria and E. coli lysates indicate that NADH is required for optimal activity. In assays with freshly prepared yeast mitochondria, the addition of NADH to 3 mM final concentration increases the rate of product formation by a factor of 2.3. However, in assays with mitochondria subjected to one freeze/thaw cycle, the addition of NADH increased methylated product formation by a factor of 2.3-6.4. The addition of NADH to such freeze/thawed samples restored levels of methyltransferase activity to those observed with freshly prepared mitochondria (data not shown). The lower level of activity in the assays with the mitochondria subjected to a freeze/thaw cycle may be due to the oxidation or loss of endogenous NADH. The methylated products were recovered in quinone form due to autoxidation. A similar dependence on NADH has been reported for the O-methylation of demethyl-Q to form Q in isolated rat liver mitochondria (53). Reduction of the demethyl-Q substrate to the hydroquinone (demethyl-QH2) eliminated the requirement for NADH in the O-methylation reaction. It is likely that the NADH is serving a similar function in the C-methylation assays and is acting to reduce the oxidized DDMQ substrate to the hydroquinone form (DDMQH2), which presumably acts as the methyl acceptor.
To determine the localization of Coq5p, a fusion gene of COQ5 and a sequence coding for the carboxyl-terminal biotinylation site of a bacterial transcarboxylase was constructed. This fusion gene complements the growth deficiency of the coq5 mutant on glycerol, indicating that the fusion protein retains activity. The detection of the fusion protein in isolated mitochondria suggests that Coq5p is located in the mitochondria. In addition, the amino terminus of Coq5p is rich in positively charged and hydroxylated residues (11 of the first 30 residues are Ser, Thr, or Arg), a characteristic of leader sequences directing polypeptide import into mitochondria (46). The mitochondrial localization of Coq5p is significant because the localization of eukaryotic Q biosynthesis has been reported to occur in several different subcellular locations, including the endoplasmic reticulum, Golgi, and mitochondria (54-57).
The ability of E. coli ubiE to rescue a yeast mutant harboring the coq5-2 allele was evaluated. Transformation of this strain with a single copy plasmid containing E. coli ubiE fused to an amino-terminal mitochondrial leader sequence and expressed from the yeast CYC1 promoter restored growth on glycerol (data not shown). This same CYC1-mitochondrial leader-ubiE construct also rescued the E. coli ubiE mutant strain, AN70 (25). The functional complementation of the yeast coq5 mutant by ubiE provides another line of evidence that these two gene products have the same function. Such interspecific functional complementation of the yeast coq mutants has been observed previously. For example, analogous constructs prepared with ubiG rescued the coq3 yeast mutant (45), expression of the E. coli polyprenyl diphosphate synthase gene rescued the yeast coq1 mutant (52), and expression of the rat COQ3 and COQ7 cDNAs rescued the corresponding yeast coq mutants (50, 51).
The availability of the yeast coq mutants provides the basis for the isolation and characterization of the COQ genes and their products in both yeast and mammals. The generation of synthetic analogs of Q intermediates provides reagents that serve as substrates for in vitro assays of enzyme activities. This approach as used in the current study shows that both E. coli UbiE and yeast Coq5 polypeptides are required for the C-methylation step in Q biosynthesis. The extent to which these gene products can independently act as C-methyltransferases is currently under investigation.
We thank Dr. Ian Young for the gift of the E. coli mutant strain AN70 and Wayne Poon, Adam Hsu, Tanya Jonassen, and Beth Marbois for helpful discussions.