From the Department of Applied Bioscience and
Biotechnology, Faculty of Life and Environmental Science and the
§ Research Institute of Molecular Genetics, Shimane
University, 1060 Nishikawatsu, Matsue 690-8504, Japan
Received for publication, August 16, 2000, and in revised form, November 2, 2000
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ABSTRACT |
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Ubiquinone (Q), composed of a quinone core
and an isoprenoid side chain, is a key component of the respiratory
chain and is an important antioxidant. In Escherichia coli,
the side chain of Q-8 is synthesized by octaprenyl-diphosphate
synthase, which is encoded by an essential gene, ispB. To
determine how IspB regulates the length of the isoprenoid, we
constructed 15 ispB mutants and expressed them in
E. coli and Saccharomyces cerevisiae. The Y38A and R321V mutants produced Q-6 and Q-7, and the Y38A/R321V double mutant produced Q-5 and Q-6, indicating that these residues are involved in the determination of chain length. E. coli
cells (ispB::cat) harboring an Arg-321 mutant
were temperature-sensitive for growth, which indicates that Arg-321 is
important for thermostability of IspB. Intriguingly, E. coli cells harboring wild-type ispB and the A79Y
mutant produced mainly Q-6, although the activity of the enzyme with
the A79Y mutation was completely abolished. When a heterodimer of
His-tagged wild-type IspB and glutathione S-transferase-tagged IspB(A79Y) was formed, the enzyme
produced a shorter length isoprenoid. These results indicate that
although the A79Y mutant is functionally inactive, it can regulate
activity upon forming a heterodimer with wild-type IspB, and this dimer formation is important for the determination of the isoprenoid chain length.
Ubiquinone (Q)1 is an
essential component of the electron transport system associated with
aerobic growth and oxidative phosphorylation in many organisms. It also
has been reported that ubiquinone has an important role as an
antioxidant in Escherichia coli (1), Schizosaccharomyces pombe (2, 38), Saccharomyces
cerevisiae (3), and mammalian cells (4). Furthermore, it was
elegantly shown that ubiquinone (or menaquinone) accepts electrons
generated by the formation of protein disulfide in E. coli
(5). Thus, multiple functions of ubiquinone have been proposed. The
ubiquinone biosynthetic pathway, which is comprised of 10 steps
including methylation, decarboxylation, hydroxylation, and isoprenoid
transfer, has been studied genetically in respiratory-deficient mutants of E. coli and S. cerevisiae (6). Each of these
organisms has a specific isoprenoid side chain length as part of the
ubiquinone molecule, e.g. Q-6 for S. cerevisiae,
Q-8 for E. coli, Q-9 for rat, and Q-10 for S. pombe and human. For this reason, ubiquinone species have been
used for classification in microbial taxonomy (7). The length of the
side chain of ubiquinone is precisely defined by the action of
polyprenyl-diphosphate synthases, but not by
4-hydroxybenzoate-polyprenyl-diphosphate transferases, which catalyze
the condensation of 4-hydroxybenzoate and polyprenyl diphosphate (8).
When various polyprenyl-diphosphate synthase genes, such as the mutant
GGPP synthase gene from Sulfolobus acidocaldarius, the
hexaprenyl-diphosphate synthase gene (COQ1) from S. cerevisiae, the heptaprenyl-diphosphate synthase gene from
Haemophilus influenzae, the octaprenyl-diphosphate synthase
gene (ispB) from E. coli, the
solanesyl-diphosphate synthase gene (sdsA) from
Rhodobacter capsulatus, and the decaprenyl-diphosphate
synthase gene (ddsA) from Gluconobacter
suboxydans, are expressed in an S. cerevisiae COQ1
mutant, each transformant produced mainly Q-5, -6, -7, -8, -9, and -10, respectively (9). When COQ2, which encodes
4-hydroxybenzoate-hexaprenyl-diphosphate transferase in S. cerevisiae, was expressed in an E. coli ubiA mutant
cell line, the transformant produce Q-8, but not Q-6 (10). These
results indicate that prenyl-diphosphate synthase determines the chain
length of ubiquinone and that 4-hydroxybenzoate-polyprenyltransferases can accept the various isoprenoid chains as a substrate.
In E. coli, ispB is an essential gene,
responsible for the biosynthesis of both ubiquinone and menaquinone
(11, 12). An E. coli ubiA mutant, which does not produce
Q-8, is not able to grow on a non-fermentable carbon source, but can
grow on glucose (10). However, an E. coli ubiA Long-chain polyprenyl-diphosphate synthases (C40,
C45, and C50) catalyze the condensation of FPP,
which acts as a primer, and IPP to produce each prenyl diphosphate with
various chain lengths. These enzymes possess seven conserved regions
including two DDXXD motifs that are binding sites for the
substrates in association with Mg2+ (13, 14). Short-chain
polyprenyl-diphosphate synthases (C15 and C20),
such as FPP and GGPP synthases, have been identified in organisms
ranging from bacteria to mammals (15), and the mechanisms that
determine the chain length have been reported (16). Site-directed
mutagenesis of farnesyl-diphosphate synthases from Bacillus
stearothermophilus (16) and certain avian species (17) and
geranylgeranyl-diphosphate synthase from S. acidocaldarius (16) was used to determine which amino acids are important for the
determination of chain length of short-chain prenyl diphosphates. These
amino acids were those at the fourth and fifth positions before an
aspartate-rich motif in region II or one amino acid at the fifth
position before this motif and two amino acids in region II.
Recently, we reported that substitution of glycine for alanine before
the first DDXXD motif in decaprenyl-diphosphate synthase
allowed the enzyme to synthesize products with longer chain lengths
(18). Thus, the fifth amino acid before region II of long-chain
polyprenyl-diphosphate synthases plays an important role in the
mechanism of chain length determination (18).
Generally, polyprenyl-diphosphate synthases are known to function as a
dimer. The medium-chain polyprenyl-diphosphate synthases (C30 and C35) from Micrococcus
luteus BP26, B. stearothermophilus, and Bacillus
subtilis are composed of heterodimers (19, 20). GGPP synthase
purified from bovine brain forms a homo-oligomer (150-195 kDa) (21).
However, the subunit structure of long-chain polyprenyl-diphosphate
synthases remains to be determined. Recently, geranyl-diphosphate
synthase isolated from spearmint (22) was found to form a heterodimer.
One subunit has similarity with known prenyltransferases, and the other
has similarity with the Arabidopsis GGR protein (23),
but the aspartate-rich motifs are not conserved.
In this study, we describe the mutational analysis of
octaprenyl-diphosphate synthase (IspB) from E. coli.
From the analysis, we found that IspB forms a homodimer that is
important for the determination of isoprenoid chain length.
Materials--
Restriction enzymes and other DNA-modifying
enzymes were purchased from Takara Shuzo Co., Ltd., and New England
Biolabs, Inc. IPP, (E)-farnesyl diphosphate
(all-(E)-FPP), geranylgeraniol, and solanesol
(all-(E)-nonaprenol) were purchased from Sigma. [1-14C]IPP (1.96 TBq/mol) was purchased from Amersham
Pharmacia Biotech. Kieselgel 60 F254 TLC plates were
purchased from Merck. Reversed-phase LKC-18 TLC plates were purchased
from Whatman.
Strains and Plasmids--
E. coli strains DH10B and
JM109 were used in the general construction of plasmids (24). KO229
(ispB::cat) (11), which is the
ispB-defective mutant of E. coli harboring pKA3
(ispB), was used as a host strain to express IspB mutants
and for ubiquinone extraction. YKK6 (COQ1::URA3)
(8), which is the COQ1-defective mutant of S. cerevisiae, was used for complementation analysis and ubiquinone
extraction. The plasmids pBluescript KS( Construction of IspB Mutants by Site-directed
Mutagenesis--
Site-directed mutagenesis by PCR was performed
following the method of Ito et al. (26). Four
oligonucleotide primers (MUT, R1 (for each mutational primer), T7, and
T3) (see Table II) were used in amplifications. pKO56, which contains
the open reading frame and downstream region of ispB, was
used as template in PCRs. First, PCR was performed with the MUT and T3
primers in one reaction and with the R1 and T7 primers in another. An
aliquot of each of the reaction mixtures was mixed in a new tube to
form the heteroduplex ispB template, and full-length mutant
ispB was amplified with the T7 and T3 primers by PCR. This
DNA fragment was digested with EcoRI and HindIII
and cloned into pBluescript KS( Complementation by Mutant ispB in an E. coli ispB Disruption
Mutant and S. cerevisiae COQ1-defective Mutant--
E. coli
KO229 (ispB::cat) harboring pKA3 (11) was
transformed with the plasmid containing mutant ispB, which
produced transformants that were resistant to spectinomycin and
ampicillin. The transformants were subcultured five times in LB medium
containing 50 µg/ml ampicillin and plated on LB agar medium
containing ampicillin. The resulting colonies were then replicated on
LB medium containing ampicillin or spectinomycin.
Spectinomycin-sensitive and ampicillin-resistant strains, which had the
mutant ispB plasmid, but not pKA3, were selected and used
for ubiquinone analysis.
To express the mutant ispB genes in S. cerevisiae
YKK6 (COQ1::URA3), a
COQ1-ispB fusion gene was constructed. The S2 and
A3 primers were used to amplify the ispB gene by PCR. The
fragment was digested with EcoRI and HindIII and
cloned into pSA1 (8), which has 53 amino acids of the Coq1
mitochondrial import signal with the COQ1 promoter. The
BamHI-HindIII fragment also was cloned into the
yeast shuttle vector YEp13M4 (25). YKK6 was transformed with both
plasmids by the lithium acetate method (27) and was selected on
Synthetic Complete (0.67% (w/v) yeast nitrogen base, 2% (w/v)
glucose or 3% (w/v) glycerol, and the appropriate amino acids) Purification of IspB--
To overexpress and purify IspB,
vectors containing the 6-His or glutathione S-transferase
(GST) tag fused to IspB were constructed. The amplified
BamHI-HindIII fragment containing ispB
from pKO56 was cloned into pQE31 (QIAGEN Inc.) to yield pQKO56. The
amplified BamHI-XhoI fragment containing
wild-type or A79Y mutant ispB was cloned into pGEX-1X in
which an XhoI linker had been inserted to yield pGKO56 or
pG79Y, respectively. The plasmids were transformed into E. coli JM109. Transformants were grown to stationary phase in LB
medium containing 50 µg/ml ampicillin, and 10 ml of culture was
inoculated into 100 ml of the same medium. The culture was grown at
37 °C for 3 h, and recombinant protein expression was induced
with 0.1 mM
isopropyl-1-thio- Coexpression of Wild-type and Mutant IspB Proteins--
The
EcoRI-HindIII fragment from pQKO56 was recloned
into pSTVK28, which had been converted from expressing chloramphenicol resistance to kanamycin resistance, to yield pSTVKQKO56. KO229 cells
harboring pBRA, which expresses IspB containing the mutation R321A,
were transformed with pSTVKQKO56 and produced transformants that were
resistant to ampicillin and kanamycin. Ampicillin-sensitive and
kanamycin-resistant strains were selected following the method described above and transformed with pG79Y, and the strains harboring both plasmids were selected and named KO229/35-2.
Ubiquinone Extraction and Measurement--
Ubiquinone extraction
was performed by the method described previously (8, 28). The crude
extract of ubiquinone was analyzed by normal-phase TLC with authentic
standard Q-10. Normal-phase TLC was carried out on a Kieselgel 60 F254 plate with benzene/acetone (97:3, v/v). The band
containing ubiquinone was collected from the TLC plate following UV
visualization and extracted with chloroform/methanol (1:1, v/v).
Samples were dried and redissolved in ethanol. The purified ubiquinone
was further analyzed by HPLC with ethanol as the solvent.
Prenyl-diphosphate Synthase Assay of Mutant IspB and Product
Analysis--
Prenyl-diphosphate synthase activity was measured by the
method described previously (8), in which incorporation of
[1-14C]IPP into reaction products is detected. E. coli DH10B or KO229 cells, harboring plasmids containing mutant
ispB, were incubated to a late log phase in LB medium
containing appropriate antibiotics at 37 °C. Cells were harvested by
centrifugation; suspended in buffer A (100 mM potassium
phosphate (pH 7.4), 5 mM EDTA, and 1 mM
2-mercaptoethanol); and ruptured by six sonication treatments, each
lasting 30 s with 30-s intervals, in an ice bath. After
centrifugation of the homogenate, the supernatant was used as a crude
enzyme extract. The assay reaction mixture contained 1.0 mM
MgCl2, 0.1% (w/v) Triton X-100, 50 mM
potassium phosphate buffer (pH 7.5), 10 µM
[1-14C]IPP (specific activity of 0.92 TBq/mol), 5 µM FPP, and 200 µg of crude extract containing the
enzyme in a final volume of 0.4 ml. Sample mixtures were incubated for
60 min at 30 °C. Reaction products such as prenyl diphosphates were
extracted with 1-butanol-saturated water and hydrolyzed with acid
phosphatase (29). The products of hydrolysis were extracted with hexane
and analyzed by reversed-phase TLC with acetone/water (19:1, v/v).
Radioactivity on the plate was detected with a BAS1500-Mac imaging
analyzer (Fuji Film Co.). The plate was exposed to iodine vapor to
detect the spots of the marker prenols.
Site-directed Mutagenesis of IspB--
It is known that the side
chain length of ubiquinone is determined by the corresponding
polyprenyl-diphosphate synthase (8), but it is not clear how
polyprenyl-diphosphate synthase determines this length. To understand
the nature of polyprenyl-diphosphate synthase, we analyzed the activity
of octaprenyl-diphosphate synthase (IspB), which produces the side
chain of Q-8 in E. coli. For this purpose, we constructed 15 IspB mutants
by site-directed mutagenesis as shown in Fig. 1 and Table
II (primers that were used).
Site-directed mutagenesis was performed following the method of Ito
et al. (26), and the substitutions in all mutant
ispB genes were confirmed by sequence analysis.
Complementation of E. coli ispB- and S. cerevisiae COQ1-defective
Mutants with Mutant ispB Genes--
E. coli DH10B was
transformed with the plasmids containing mutant ispB genes,
and the transformants were used in ubiquinone analysis. Because DH10B
has the wild-type ispB gene in the form of genomic DNA, the
main product is expected to be Q-8. Although most DH10B cells harboring
the mutant ispB gene produced Q-8, a number of mutants
produced Q-8 with small amounts of Q-6 and Q-7 (data not shown); and
interestingly, DH10B harboring the A79Y mutant produced mainly Q-6 (see
Fig. 6A). To detect the actual ubiquinone species produced
by the product of the mutant ispB gene, E. coli
KO229 (ispB::cat)/pKA3 (ispB), which is
defective for the genomic ispB gene, but retains
ispB in a plasmid, was transformed with the plasmids
containing the mutant ispB genes. KO229, which harbors the
mutant ispB genes and has lost the wild-type ispB
plasmid (pKA3), was selected as described under "Experimental Procedures." L31V, I32V, Y38A, Y37A/Y38A, Y38A/R321V, Y61V, F75A, K235L, R321A, R321D, and R321V mutant KO229 strains were obtained; however, Y37A, A79Y, K170G, and K170A mutant KO229 strains could not be
isolated. Since ispB is essential for growth of E. coli (11), the inability to replace wild-type ispB with
these mutants suggested that the Y37A, A79Y, K170G, and K170A mutants
do not retain functional activity. The mutants that could complement the loss of the wild-type gene were further analyzed by ubiquinone extraction and analysis (Fig. 2). In the
Y38A mutant, Q-7 was mainly produced, with lesser amounts of Q-6 and
Q-8 (Fig. 2D). In the Y37A/Y38A mutant, Q-7 and Q-6 were
mainly produced, with a little Q-8 (Fig. 2E). In the
Y38A/R321V and R321V mutants, Q-6 was mainly produced, with a small
amount of Q-5 and Q-7 (Fig. 2, F and L,
respectively); however, hardly any Q-8 was produced. In the K235L and
R321A mutants, Q-8 was mainly produced; however, a minor product (Q-7)
was produced at a level that was greater than that with wild-type IspB
(Fig. 2, I and J, respectively). These results
indicate that Tyr-38, Lys-235, and Arg-321 are involved in chain length
determination.
Although ispB in E. coli is essential for growth,
the chromosomal COQ1 gene (homolog of ispB) in
S. cerevisiae can be deleted to produce a
respiration-deficient phenotype. We took advantage of this
COQ1 mutant phenotype for analysis of the function of ispB (8). To express ispB mutants and to analyze
their ubiquinone production in YKK6 (COQ1::URA3),
mutant ispB genes fused with 53 amino acids of the Coq1
mitochondrial import signal were constructed. YKK6 was transformed with
various mutant ispB fusion plasmids, and transformants were
replicated on Synthetic Complete
We next analyzed the ubiquinone species produced by YKK6 harboring
mutant ispB plasmids (Fig. 4).
In the Y38A mutant, Q-8 was mainly produced, along with a significant
amount of Q-7 (Fig. 4C). In the Y37A/Y38A mutant, Q-8 and
Q-7 were mainly produced, with a lesser amount of Q-6 and Q-5 (Fig.
4D). In the R321V mutant, three ubiquinone species (Q-7,
Q-6, and Q-5) were mainly produced, with a small amount of Q-8 (Fig.
4I). In the Y38A/R321V mutant, Q-5 was the main product,
with lesser amounts of Q-6 to Q-8 (Fig. 4E). These results
again suggest that Tyr-38 and Arg-321 are associated with chain length
determination. Most species of ubiquinone produced by mutant
ispB in E. coli were similar to ones produced in
S. cerevisiae, but some ubiquinone species were shorter in
length in S. cerevisiae compared with the ones produced in
E. coli.
Arg-321 Is Important for Thermostability of IspB--
We tested
all ispB mutants for temperature sensitivity and found two
temperature-sensitive mutants. The KO229 strain harboring the Arg-321
mutant (R321A or R321D) grew on LB medium containing chloramphenicol
and ampicillin at 30 °C (Fig.
5A, a), but
stopped growing or grew only slowly at 43 °C (Fig. 5A,
b), whereas KO229/pKA3 grew normally at 43 °C. The growth
curves of the mutants are shown in Fig. 5B. The R321A,
R321D, and R321V mutant KO229 strains grew normally at 30 °C; but
the R321A and R321D mutant KO229 strains did not grow at all at
43 °C, and the R321V mutant KO229 strain grew slowly and reached a
plateau phase faster than KO229/pKA3 at 43 °C. These results
indicate that Arg-321 is important for thermostability of IspB, as well
as reconfirm that ispB is essential for the growth of
E. coli.
Overexpression, Purification, and Characterization of IspB--
As
mentioned above, we constructed the 15 IspB mutants by site-directed
mutagenesis and expressed them in E. coli and S. cerevisiae. Among these mutants, the A79Y mutant had an
interesting property in that DH10B harboring the A79Y mutant produced
Q-6 (Fig. 6A), whereas the
A79Y mutant in S. cerevisiae YKK6
(COQ1::URA3) did not retain functional activity
(Fig. 3). To further analyze the A79Y mutant, we purified the wild-type
and A79Y mutant IspB proteins. JM109 harboring pQKO56 produced His-IspB
fusion proteins with a 6-His tag at the amino terminus of wild-type
IspB. JM109 harboring pGKO56 and pG79Y produced GST-IspB and
GST-IspB(A79Y) fusion proteins, respectively, with GST at the amino
terminus. His-IspB, GST-IspB, and GST-IspB(A79Y) were purified, and
prenyltransferase activity was examined as described under
"Experimental Procedures." Purified His-IspB and GST-IspB retained
functional activity of octaprenyl-diphosphate synthase, but
GST-IspB(A79Y) had no detectable activity (data not shown). This result
suggests that the substitution of tyrosine for alanine at position 79 abolishes the enzyme activity of IspB, which is consistent with the
results of A79Y mutant activity in YKK6 (Fig. 3).
Wild-type IspB and IspB(A79Y) Form a Dimer Structure--
We next
tested, by Western blot analysis, whether wild-type IspB and IspB(A79Y)
form dimers. Purified His-IspB and His-DdsA (as a negative control)
were incubated with GST-IspB(A79Y) in buffer A and then subjected
to Ni2+-NTA (Fig. 6C, lanes 2) or
glutathione-Sepharose 4B (lane 5) column chromatography. The
His-IspB protein was detected together with GST-IspB(A79Y) from
purified products on the glutathione-Sepharose 4B column (Fig.
6C, lanes 5), but the His-DdsA protein was not detected in the GST-purified products (Fig. 6C, panel
a, lane 6). Conversely, GST-IspB(A79Y) was detected
with His-IspB from purified proteins separated on the
Ni2+-NTA column (Fig. 6C, panel b,
lane 2). Homodimerization of wild-type IspB and the A79Y
mutant itself was also observed. When purified His-IspB was incubated
with GST-IspB(A79Y) in buffer A with disuccinimidyl suberate as a
protein cross-linker and subjected to electrophoresis, the band
corresponding to a heterodimer with a molecular mass of 97 kDa was
detected (data not shown). These results indicate that wild-type IspB
and IspB(A79Y) can form a heterodimer, but these proteins cannot form
dimers with the similarly structured enzyme DdsA (18). We analyzed the
chain lengths of products of heterodimeric IspB consisting of wild-type
and A79Y subunits in vitro. Only octaprenyl diphosphate was
detected in this assay. We believe that because the homodimer of
His-IspB comprises the majority of active enzyme in
vitro, we could not detect the activity of the heterodimer
composed of IspB and IspB(A79Y). We then made a strain expressing both
His-IspB and GST-IspB(A79Y). We constructed the pSTVKQKO56 plasmid,
which expresses His-IspB, and co-transformed it with the pG79Y plasmid,
which expresses GST-IspB(A79Y); and the resulting strain was named
KO229/35-2. This strain produced Q-6 and Q-8 (Fig. 6B).
Crude proteins were extracted from KO229/35-2 and purified on a
Ni2+-NTA column (Fig. 6C, lanes 1) or
a glutathione-Sepharose 4B column (lanes 4). The His-IspB
protein was detected in the GST-tagged purified proteins (Fig.
6C, panel a, lane 4); and similarly,
the GST-IspB(A79Y) protein was detected in the His-tagged
purified proteins (panel b, lane 1). Thus, it
was confirmed that a heterodimer of wild-type IspB and IspB(A79Y)
formed in KO229/35-2. When enzyme activity analysis was performed again
using crude extract from KO229/35-2, isoprenoid products shorter than
octaprenyl diphosphate were detected (Fig.
7, lane 3), although in KO229
harboring only pSTVKQKO56, octaprenyl diphosphate alone was produced
(lane 2). These results indicate that the IspB(A79Y) mutant
can function as a component of heterodimeric IspB to produce shorter
isoprenoid chains compared with the isoprenoid product of homodimeric
IspB, although IspB(A79Y) itself does not retain enzyme activity.
In E. coli, the side chain length of Q-8 is determined
by octaprenyl-diphosphate synthase (IspB) (8). To discover how IspB determines the chain length, we constructed 15 IspB mutants and expressed them in the E. coli ispB-defective mutant KO229
and in the S. cerevisiae COQ1-defective mutant YKK6 and then
analyzed the ubiquinone species produced. In KO229 or YKK6 expressing
the IspB mutants L31V, I32V, Y61V, and F75A, the resulting ubiquinone species were almost the same as those produced by the wild-type enzyme
(Figs. 2 and 4). However, in KO229 cells expressing the IspB mutant
Y38A, Q-7 was the major product; and in KO229 cells expressing the
Y37A/Y38A double mutant, the levels of Q-6 were increased. Although the
Tyr-37 mutant of IspB did not have enzyme activity, an additional
mutation at Tyr-38 restored activity. We speculate that the abnormal
protein structure in the Tyr-37 mutant is compensated by an additional
Tyr-38 mutation. In E. coli KO229/pKA3 cells,
complementation of the lost ispB gene that resided on a
plasmid with the Y37A, A79Y, K170G, and K170A mutant ispB
genes was unsuccessful. Furthermore, S. cerevisiae YKK6
transformed with the same genes could not grow on Synthetic
Complete Lys-170 is conserved in all known polyprenyl-diphosphate synthases from
various organisms. Mutation of the corresponding lysine to glutamic
acid in farnesyl-diphosphate synthase from S. cerevisiae produced an enzyme that synthesized geranyl diphosphate rather than FPP
because the glutamic acid substitution decreased substrate affinity
(32). The substitution of this corresponding lysine in IspB with
alanine or glycine strongly decreased enzyme activity.
KO229 harboring the Arg-321 mutant (R321A or R321D) grew at 30 °C,
but did not grow at 43 °C (Fig. 5). The arginine located at the
third position from the carboxyl terminus is conserved in all
long-chain polyprenyl-diphosphate synthases (Fig. 1) and FPP synthases
(14). However, in GGPP synthases, there is very little conservation in
the C-terminal region; for example, the corresponding amino acid in
mouse and human GGPP synthases is glutamate (15). Amino acid alignment
of GGPP synthases showed that the length of the carboxyl terminus
region was different between FPP synthases and long-chain
polyprenyl-diphosphate synthases. It has been reported that an FPP
synthase mutant from B. stearothermophilus in which the
arginine was replaced by valine had catalytic activity similar to that
of the wild-type enzyme, indicating that the amino acids in the
carboxyl terminus are not essential for catalytic function in FPP
synthase (33). These results suggest that the role of the carboxyl
terminus in FPP and GGPP synthases is different from that in long-chain
polyprenyl-diphosphate synthases. In the IspB(R321V) mutant, the
ubiquinone product had a shorter isoprenoid side chain, and
KO229 cells carrying the ispB gene with this mutation did not grow at 43 °C. These results suggest that Arg-321 is
important for thermostability of IspB. This temperature-sensitive
Arg-321 mutant will be very useful for future study to determine why
ispB is an essential gene in E. coli.
Ala-79 of IspB is the fifth amino acid before the first aspartate-rich
motif. This position is important for chain length determination by FPP
synthases (16) and decaprenyl-diphosphate synthase (18). In FPP
synthases, the corresponding residue is an aromatic amino acid, either
tyrosine or phenylalanine. When this amino acid residue is substituted
with glycine, an isoprenoid longer than the wild-type product is
produced (16). Similarly, substitution of the corresponding amino acid
(G70Y) in DdsA resulted in production of a small amount of undecaprenyl
diphosphate that was longer than the original decaprenyl diphosphate
(18); however, the A70Y mutant of DdsA had no activity. This is
consistent with our observation that the IspB(A79Y) mutant did not have
enzyme activity. Interestingly, wild-type E. coli DH10B
harboring the A79Y mutant produced Q-6, although the A79Y mutant itself
had no activity (Fig. 6A). We have shown that the
nonfunctional IspB(A79Y) protein forms a dimer with wild-type IspB
produced by the genomic ispB gene and synthesizes a
ubiquinone product with an altered chain length. We have also shown
that His-IspB and GST-IspB(A79Y) form a heterodimer and produce shorter
isoprenoids than octaprenyl diphosphate. As shown in a model
(Fig. 8), the homodimer of the A79Y
mutant did not have catalytic activity, but the heterodimer of
wild-type IspB and the A79Y mutant did have activity and produced a
shorter isoprenoid than a homodimer of wild-type IspB. This result
means in a sense that in the creation of IspB-IspB(A79Y), we have
produced a hexaprenyl-diphosphate synthase. Furthermore, this
IspB-IspB(A79Y) form can be thought of as an evolutional intermediate
toward a heterodimer of the medium-chain polyprenyl-diphosphate synthases, such as hexaprenyl- and heptaprenyl-diphosphate synthases from M. luteus BP26, B. stearothermophilus, and
B. subtilis (19, 20). These heterodimers are composed of
components I and II; component I does not resemble other proteins,
whereas component II has conserved regions, similar to known
polyprenyl-diphosphate synthases. Component I serves the cavity
for a catalitic space of substrate and has an important role in chain
length determination (34). IspB(A79Y) probably plays a role similar to
that of component I in a IspB-IspB(A79Y) heterodimer. In support of
this idea, it has been reported that the subunits of FPP synthase
interact with each other to form a shared active site in the homodimer
structure (35).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
menA
mutant, which lacks both ubiquinone and
menaquinone biosynthesis genes, can grow only when a small amount of
Q-8 is still produced by leakiness of the mutations. Thus,
ubiA
menA
mutants
with an absolute lack of production of Q-8 and menaquinone-8 cannot be isolated.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)/SK(+) and YEp13M4 were used
as vectors (24, 25). The strains and plasmids used in this study are
listed in Table I.
Strains and plasmids used in this study
). This construct was transformed into
E. coli DH10B and KO229 (ispB::cat) for
analysis of enzyme activity and ubiquinone production.
Leu
Ura medium.
-D-galactopyranoside. The cells were
collected by centrifugation at 2500 × g for 10 min. To
purify His-tagged IspB, cells were suspended in 50 mM
sodium phosphate, 300 mM NaCl, and 10 mM
imidazole and sonicated 10 times for 10 s at 10-s intervals with
an ultrasonic disintegrator in an ice bath. Ruptured cells were
centrifuged at 15,000 × g for 20 min. The resulting
supernatants were added to a Ni2+-nitrilotriacetic acid
(NTA) slurry and mixed gently at 4 °C for 60 min. This mixture was
loaded onto a column and washed with 50 mM sodium
phosphate, 300 mM NaCl, and 20 mM imidazole.
The His-IspB protein was eluted with 50 mM sodium
phosphate, 300 mM NaCl, and 250 mM imidazole.
To purify the GST-IspB protein, cells were suspended in 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol (sonication
buffer). Cells were ruptured by sonication, and the lysate was mixed
with glutathione-Sepharose 4B (Amersham Pharmacia Biotech) at 4 °C
for 60 min. This mixture was washed twice with 140 mM NaCl,
2.7 mM KCl, 10 mM sodium phosphate, and 1.8 mM potassium phosphate and then with sonication buffer. The
GST-IspB protein was eluted with sonication buffer containing 10 mM reduced glutathione.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Alignment of the amino acid sequences of
bacterial polyprenyl-diphosphate synthases. HIHEP,
H. influenzae heptaprenyl-diphosphate synthase
(GenBankTM/EBI accession number U32770); ECISPB,
E. coli octaprenyl-diphosphate synthase (accession number
U18997); SNSPS, Synechocystis sp. strain PCC6803
(accession number D90899); RCSDSA, R. capsulatus
solanesyl-diphosphate synthase (accession number AB001997);
GSDDSA, G. suboxydans decaprenyl-diphosphate
synthase (accession number AB006850). Conserved residues in more than
two out of five sequences are boxed. Conserved regions
(I-VII) are underlined. Triangles
indicate the corresponding mutated sites of IspB. Numbers on
the right indicate amino acid residue positions. Asterisks
indicate the end of the proteins.
Primers used to construct ispB mutants in this study
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Fig. 2.
HPLC analysis of ubiquinone extracted from
KO229 harboring various mutant ispB genes.
Ubiquinone was extracted from E. coli KO229 harboring each
ispB gene mutated as follows: A, wild-type
(w. t.) IspB; B, L31V; C, I32V;
D, Y38A; E, Y37A/Y38A; F, Y38A/R321V;
G, Y61V; H, F75A; I, K235L;
J, R321A; K, R321D; L, R321V.
Leu
Ura plates containing glucose
(Fig. 3A) or glycerol (Fig.
3B) as a non-fermentable carbon source. Although most of the
strains grew on the glycerol plate, the Y37A, A79Y, K170G, and K170A
mutant YKK6 strains did not grow, indicating that these mutants do not retain functional activity. These results are consistent with the
complementation analysis of mutants in E. coli KO229/pKA3 (Fig. 2).
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Fig. 3.
Complementation of respiration
deficiency in the S. cerevisiae COQ1 disruptant strain
by various mutant ispB genes. The YKK6
(COQ1::URA3) strains harboring each of the
following plasmids were tested: I, YEp13M4 vector;
II, IspB; III, R321V; IV, K170A;
V, R321A; VI, R321D; VII, L31V;
VIII, I32V; IX, Y38A; X, K235L;
XI, Y37A; XII, Y61V; XIII, A79Y;
XIV, Y37A/Y38A; XV, Y38A/R321V; XVI,
K170G. These strains were grown on Synthetic Complete Leu
Ura medium
with glucose (A) or glycerol (B) at 30 °C for
6 days.
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Fig. 4.
HPLC analysis of ubiquinone extracted from
YKK6 harboring the COQ1-ispB fusion plasmid.
Ubiquinone was extracted from S. cerevisiae YKK6 harboring
each COQ1-ispB fusion plasmid mutated as follows:
A, YEp13M4; B, wild-type (w. t.) IspB;
C, Y38A; D, Y37A/Y38A; E, Y38A/R321V;
F, Y61V; G, R321A; H, R321D;
I, R321V.
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Fig. 5.
Growth of E. coli KO229
harboring Arg-321 mutant IspB. A, KO229 harboring pBRA
(a) or pKA3 (b) was grown on LB plates containing
chloramphenicol and ampicillin at 30 or 43 °C. B,
different KO229 strains harboring each plasmid were grown on LB liquid
medium containing chloramphenicol and ampicillin at 30 or 43 °C.
, pKA3;
, pBV16 (R321V);
, pBRA (R321A);
, pBRD
(R321D).
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Fig. 6.
Ubiquinone analysis and dimer formation
of wild-type IspB and IspB(A79Y). A and B,
shown are the results from the analysis of ubiquinones produced in
DH10B harboring the A79Y mutant ispB gene and in KO229
harboring pSTVKQKO56 and pG79Y, respectively, by HPLC. C,
crude proteins were extracted from KO229 harboring pSTVKQKO56 and pG79Y
(KO229/35-2 strain) and incubated in buffer A containing 1.0 mM MgCl2 and 0.1% (w/v) Triton X-100 at
30 °C for 1 h and then purified on a Ni2+-NTA
column (lanes 1) or a glutathione-Sepharose 4B column
(lanes 4). Purified His-IspB and GST-IspB(A79Y) proteins
were used in incubations as described above and purified on a
Ni2+-NTA column (lanes 2) or a
glutathione-Sepharose 4B column (lanes 5). Purified His-DdsA
and GST-IspB(A79Y) proteins were used for the same experiment and
purified on a glutathione-Sepharose 4B column (lanes 6).
Purified His-IspB (lanes 3) and GST-IspB(A79Y) (lanes
7) proteins were loaded as controls. Western blot analysis was
performed using anti-6-His antibody (panel a) or anti-GST
antibody (panel b). Arrowheads indicate the
positions of His-IspB (panel a) or GST-IspB(A79Y)
(panel b) protein. The asterisk indicates
background protein recognized by the antibody.
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Fig. 7.
Reversed-phase thin-layer chromatogram of the
products of heterodimeric IspB and IspB(A79Y). The enzyme reaction
was carried out using [1-14C]IPP and FPP as substrates
with cell extract as the crude enzyme. The reaction products were
hydrolyzed by acid phosphatase, and the resulting alcohols were
analyzed by reversed-phase TLC. The arrowheads indicate
positions as follows: GGOH,
all-(E)-geranylgeraniol as a standard; SOH,
solanesol as a standard; OOH, octaprenol; Ori.,
origin; S. F., solvent front. Lane 1,
KO229/pKA3; lane 2, KO229/pSTVKQKO56; lane 3,
KO229/pSTVKQKO56/pG79Y (KO229/35-2 strain).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Leu
Ura containing glycerol (Fig. 3). These results indicate
that Tyr-37, Ala-79, and Lys-170 are important residues for activity.
The pattern of ubiquinone species synthesized in E. coli or
S. cerevisiae harboring the same mutant IspB was some
different. This difference might be due to the difference in the
intracellular conditions of two organisms because it was reported that
metal ions and substrate concentration affected the produced chain
length catalyzed by prenyl-diphosphate synthases (30, 31). We
intentionally mutated aromatic residues located before the first
aspartate-rich motif because tyrosine or phenylalanine is important for
chain length determination in FPP and GGPP synthases (16). From our
results, we concluded that Tyr-61 and Phe-75 are not important for
catalytic activity, whereas Tyr-37 and Tyr-38 play an important role in catalytic activity and chain length determination.
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Fig. 8.
Model for dimerization of wild-type IspB and
IspB(A79Y) and their products. Wild-type IspB (W. T.) forms a homodimer to produce Q-8. The A79Y mutant forms a
nonfunctional homodimer. Wild-type IspB and IspB(A79Y) form a
heterodimer, which produces ubiquinone (UQ) with a shorter
chain length.
The formation of homodimers of long chain-producing prenyl-diphosphate
synthase was demonstrated, for the first time, in this study. In
addition, homodimerization of long chain-producing prenyl-diphosphate synthase was found to be important for enzyme activity and chain length
determination. Long-chain polyprenyl-diphosphate synthase (C40, C45, and C50) genes have been
isolated from E. coli (36), Synechocystis sp.
strain PCC6803 (11), R. capsulatus (37), G. suboxydans (18) and S. pombe (2). Because these
synthases, except for the one from S. pombe, could be
expressed in E. coli and S. cerevisiae, we
believe that they form homodimeric enzymes like IspB. However, because
the enzyme from the eukaryote was not able to express a functional
product in the other species, eukaryotic long-chain
polyprenyl-diphosphate synthase might be regulated differently.
Therefore, the long-chain polyprenyl-diphosphate synthase of eukaryotic
origin will be an interesting subject for further analysis.
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FOOTNOTES |
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* This work was supported by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan and by a Sasakawa scientific research grant from the Japan Science Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 81-852-32-6587; Fax: 81-852-32-6499; E-mail: kawamuka@life.shimane-u.ac.jp.
Published, JBC Papers in Press, December 6, 2000, DOI 10.1074/jbc.M007472200
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ABBREVIATIONS |
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The abbreviations used are: Q, ubiquinone; GGPP, geranylgeranyl diphosphate; FPP, farnesyl diphosphate; IPP, isopentenyl diphosphate; PCR, polymerase chain reaction; GST, glutathione S-transferase; NTA, nitrilotriacetic acid; HPLC, high pressure liquid chromatography.
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