(Received for publication, December 1, 1995; and in revised form, January 18, 1996)
From the
Prenyltransferases catalyze the consecutive condensation of
isopentenyl diphosphate (IPP) with allylic diphosphates to produce
prenyl diphosphates whose chain lengths are absolutely determined by
each enzyme. In order to investigate the mechanisms of the consecutive
reaction and of the determination of ultimate chain length, a random
mutational approach was planned. The farnesyl diphosphate (FPP)
synthase gene of Bacillus stearothermophilus was subjected to
random mutagenesis by NaNO treatment to construct libraries
of mutated FPP synthase genes on a high-copy plasmid. From the
libraries, the mutants that showed the activity of geranylgeranyl
diphosphate (GGPP) synthase were selected by the red-white screening
method (Ohnuma, S.-i., Suzuki, M., and Nishino, T.(1994) J. Biol.
Chem. 268, 14792-14797), which utilized carotenoid synthetic
genes, phytoene synthase, and phytoene desaturase, to visualize the
formation of GGPP in vivo. Eleven red positive clones were
identified from about 24,300 mutants, and four (mutant 1, 2, 3, and 4)
of them were analyzed for the enzyme activities. Results of in
vitro assays demonstrated that all these mutants produced
(all-E)-GGPP although the amounts were different. Each mutant
was found to contain a few amino acid substitutions: mutant 1, Y81H and
L275S; mutant 2, L34V and R59Q; mutant 3, V157A and H182Y; mutant 4,
Y81H, P239R, and A265T. Site-directed mutagenesis showed that Y81H,
L34V, or V157A was essential for the expression of the activity of GGPP
synthase. Especially, the replacement of tyrosine 81 by histidine is
the most effective because the production ratios of GGPP to FPP in
mutant 1 and 4 are the largest. Based on prediction of the secondary
structure, it is revealed that the tyrosine 81 situates on a point 11
12 Å apart from the first DDXXD motif, whose
distance is similar to the length of hydrocarbon moiety of FPP. These
data might suggest that the aromatic ring of tyrosine 81 blocks the
chain elongation longer than FPP. Comparisons of kinetic parameters of
the mutated and wild type enzymes revealed several phenomena that may
relate with the change of the ultimate chain length. They are a
decrease of the total reaction rate, increase of K
for dimethylallyl diphosphate, decrease
of V
for dimethylallyl diphosphate, and allylic
substrate dependence of K
for IPP.
Prenyltransferases catalyze the consecutive condensation of
isopentenyl diphosphate (IPP) ()with allylic diphosphates to
synthesize prenyl diphosphates with various chain lengths. These
enzymes are classified according to the chain length of the final
product and the geometry of the double bond that is formed by the
condensation. So far a number of prenyltransferases have been found
from various organisms and characterized(1) . For example, FPP
synthase, which is a key enzyme of the biosynthesis of steroids, prenyl
quinones, farnesylated protein, and dolichols, catalyzes the
condensations of IPP with DMAPP (C
) and with geranyl
diphosphate (GPP, C
) to give FPP (C
) as the
ultimate product (Fig. 1A). GGPP synthase, whose
product is a precursor of carotenoids, geranylgeranylated proteins, and
ether-linked lipids of archaebacterium, utilizes DMAPP, GPP, and FPP as
allylic substrates to give an amphiphilic molecule containing 4
isoprene units, GGPP (C
). Solanesyl diphosphate synthase
catalyzes the consecutive condensation of IPP with E-stereochemistry to produce a C
compound.
Although these enzymes catalyze similar condensation reactions, they do
not catalyze the condensation beyond the limit of the chain length of
product determined by their own specificities. Why does the
condensation stop at the step that is determined by each enzyme?
Figure 1:
Pathway of isoprenoid biosynthesis (A) and schematic diagram of the red-white screening (B). A, wild-type FPP synthase of B.
stearothermophilus catalyzes consecutive condensations of IPP to
produce FPP as an ultimate product. We selected mutated FPP synthase
that has GGPP synthase activity by using the color selection system,
which utilizes carotenoid synthetic genes (crtB and crtI). B, E. coli DH5 was transformed
with pACYC-IB, which expresses phytoene synthase (crtB) and
phytoene desaturase (crtI). The cells were transformed with
the plasmids derived from the library of random mutated FPP synthase,
plated on LB plate, and then red colonies, which mean that the mutated
FPP synthases have GGPP synthase activity, were
isolated.
The prenyl chain length of respiratory quinones is altered by viral infection and differs from tissue to tissue. The dolichyl chain length in rat liver also changes on carcinogenesis (2) or aging(3, 4) . It has also been reported that the product chain length of prenyltransferases is changed under some reaction conditions(5, 6, 7, 8) . In all cases, the chain length always changes shorter than the ultimate chain length. Ohnuma et al.(9) and Matsuoka et al.(10) have suggested on the basis of in vitro examinations that these phenomena reflect the level of IPP and metal ions in the living cells. However, from these lines of evidence, it is difficult to understand the mechanisms that force each prenyltransferase to yield its intrinsic product.
During the past few years the amino acid sequences of FPP synthases (11, 12, 13, 14, 15) , GGPP synthases(16, 17, 18, 19, 20) , hexaprenyl diphosphate synthase (21) , heptaprenyl diphosphate synthase(22) , and octaprenyl diphosphate synthase (23) have been determined. Comparisons of the primary structures revealed several conserved domains including two aspartate-rich domains, DDXXD, where X encodes any amino acid. In FPP synthase, site-directed mutagenesis studies have been carried out by several groups (23, 24, 25, 26, 27) with special attention to the two aspartate-rich domains. These studies have indicated that the aspartate-rich domains are essential for catalytic activity. It is suggested that the aspartate residues bind the diphosphate moieties of IPP and allylic substrate through a magnesium bridge. However, none of the previous studies answered the question which amino acid residues are important in determining the chain length of the ultimate product.
In order to obtain information about amino
acid residues that are related to chain-length determination, we tried
to convert FPP synthase to GGPP synthase using random chemical
mutagenesis. If combined with biological selection, the random
mutagenesis provides a powerful method for identifying important amino
acid residues. Recently, we have developed an in vivo method
for detecting GGPP synthase activity, which utilizes carotenoid
biosynthesis genes of Erwinia uredovora to visualize a colored
clone expressing GGPP synthase activity(19) . We introduced
random mutations on the Bacillus stearothermophilus FPP
synthase gene using NaNO, and screened the clones that
showed GGPP synthase activity by taking advantage of the color
selection method (Fig. 1). This paper reports the determination
of the amino acid residues that are important for chain length
determination.
In order to screen the mutants having GGPP
synthase activity, the red-white screening system reported previously (19) was used (Fig. 1). In this system, if the GGPP
synthase activity is expressed in the transformant E. coli cell that expresses the genes for phytoene synthase and phytoene
desaturase, the transformant should produce lycopene and become red.
Before screening, we confirmed the background level of GGPP synthase
activity of wild-type B. stearothermophilus FPP synthase
because avian liver FPP synthase is known to have a weak GGPP synthase
activity(32) . E. coli DH5 containing pACYC-IB
was transformed with pEX11 or pFPS, which expressed wild-type FPP
synthase. The color of both transformed cells remained white. These
data showed that the GGPP synthase activity of wild-type FPP synthase
was, if any, negligible in vivo. Then, cells carrying pACYC-IB
were transformed with both plasmids derived from the libraries of 0.25
and 1 M NaNO
treatments. Approximately 16,700 and
7,600 recombinants from both libraries were screened. As a result, 1
and 10 red colonies were obtained, respectively. Four positive colonies
were isolated from the library derived from 1 M NaNO
treatment, and then the pTV118N derivatives (pMU1, pMU2, pMU3,
and pMU4) were isolated from the clones.
Figure 2:
TLC autoradiochromatograms of the alcohols
obtained by enzymatic hydrolysis of the products formed by the mutated
FPP synthase. Panels A and B, the sample from
incubation of [1-C]IPP and FPP with the
indicated pure enzyme was analyzed by reversed phase LKC-18 TLC (A) and normal phase Kieselgel 60 TLC (B) as
described under ``Experimental Procedures.'' Panel
C, the sample from incubation of [1-
C]IPP
and GPP with the indicated pure enzyme was analyzed by reversed phase
LKC-18 TLC. Panel D, the sample from incubation of
[1-
C]IPP and DMAPP with the indicated pure
enzyme was analyzed by reversed phase LKC-18 TLC. Spots of authentic
standard alcohols: GOH, geraniol; FOH, farnesol; GGOH, geranylgeraniol. Ori., origin; S.F.,
solvent front.
In the case
of mutant 1, the K for DMAPP was larger than that
of wild-type, and the V
was much smaller than
that of wild-type, indicating that it is difficult for mutant 1 to
accept DMAPP as a primer substrate. The K
for GPP
also increased, but the decrease of V
was less
marked than that for DMAPP. GPP was a better substrate than DMAPP. The K
for FPP was the smallest among the three allylic
substrates and V
/K
for FPP
was the largest. These data show that the activity of allylic
substrates increases in the order of DMAPP, GPP, and FPP. The
observation in the product analysis of mutant 1 that the amounts of GPP
and FPP were smaller than that of GGPP (Fig. 2, C, lane 1 and D, lane 1) seems to reflect the activities of the
allylic substrates. The kinetic constants for IPP were independently
determined using DMAPP, GPP, and FPP as countersubstrates. The K
values for IPP were 265 µM (countersubstrate, DMAPP), 12.4 µM (GPP), and 9.40
µM (FPP). The values were dramatically different depending
on the allylic substrate to be used as a co-substrate. These data
suggest that the affinity of the enzyme to IPP depends upon the
structure of allylic substrate. The K
values and V
/K
for IPP also indicate
that this mutation alters the catalytic properties so that the enzyme
accepts IPP and FPP as preferable substrates.
In the case of mutant
2, the K for DMAPP became larger than that of
wild-type, whereas the K
of GPP decreased. These
results indicate that the affinity for allylic substrates slightly
changes in such a way that the enzyme prefers GPP to DMAPP. However,
the K
for FPP is larger than that of GPP, which is
different from the case of mutant 1. The V
for
FPP was smaller than that for GPP or that for FPP in mutant 1.
Therefore, the formation of GGPP by mutant 2 seems to be less than that
of FPP (Fig. 2, C, lane 2 and D, lane 2). The K
for IPP was also dependent on the allylic
substrate employed, although the dependence was less marked than that
of mutant 1. Mutant 2 shows a smaller K
value for
GPP than for DMAPP or FPP. In addition, the K
value for IPP is the smallest when the co-substrate is GPP. These
results indicate that the combination of IPP and GPP is the most
acceptable for this mutant enzyme.
In the case of mutant 3, the K for DMAPP was greater than that of wild-type,
and the V
for DMAPP was smaller than that of
wild-type. The K
for GPP was slightly smaller than
that of wild-type and the smallest among those of the allylic
substrates. In comparison with mutant 2, the K
values for both allylic substrates and IPP were greater than
those of mutant 2 except for the K
value of IPP in
the reaction with GPP. When GPP was used, the K
of
IPP was the smallest among all cases.
The profile of K values for both allylic substrates and IPP in
mutant 4 was similar to that in mutant 1. As pointed out in the
following paragraph, there is the same amino acid substitution in both
mutants 1 and 4. Therefore, it seems reasonable to conclude that this
substitution is essential for the formation of GGPP. However, most of
the V
values of mutant 4 were lower than those
of mutant 1, whereas most of the K
values of
mutant 4 were lower than those of mutant 1. Although the different
substitution in mutants 1 and 4 brought about the change in kinetic
properties, it caused no significant change in product distribution.
In experiments described above, we determined the amino acid substitutions that are required for GGPP synthase activity in mutants 1 and 4. Next, we tried to determine the essential amino acid of mutations 2 and 3. The four recombinants, L34V, R59Q, V157A, and H182Y, which each contained a single amino acid substitution, were made by the Kunkel method, and then E. coli harboring pACYC-IB was transformed with each of the recombinants. Two (L34V, V157A) of the four mutants produced red colonies and the other made white colonies (Table 5). However, the red color of the colonies derived from L34V was lighter than that of pMU2. Hence, replacement of Arg-59 with Gln, which was also observed in mutant 2, might contribute to the change in the chain length of the product. These observations clearly show that single amino acid substitutions alter the ultimate chain length of the product that is determined intrinsically by individual prenyltransferase.
We tried to convert FPP synthase to GGPP synthase by using random mutagenesis and phenotypic screening. An essential element of this strategy is the efficient identification of mutants of interest among a large population of variants. For this purpose we used the red-white screening system that utilized phytoene synthase (crtB) and phytoene desaturase (crtI) to visualize the formation of GGPP in vivo. Eleven mutants that formed red colonies were selected from 24,300 clones. Four of them were analyzed in detail by sequencing the gene and determining the essential amino acid residues, reaction products, and kinetic constants. These mutants showed different properties. Characteristics of each mutant are summarized below.
Mutant 1 contained two amino acid alterations
(Y81H, L275S). Y81H was found to be essential for expression of GGPP
synthase activity. This enzyme mainly produced GGPP when any of the
allylic substrates were used as a priming substrate, and it showed the
highest GGPP synthase activity among the four mutants. Both of the K values for DMAPP and GPP were increased as
compared with those of wild-type. Especially, the increase of K
for DMAPP was prominent. The Michaelis constant
for allylic substrate became smaller as the chain length of the allylic
substrate increased. Consequently, FPP showed a higher affinity than
DMAPP or GPP. The V
for DMAPP and GPP dropped.
The K
values for IPP depended on the primer
substrate employed, being decreased as the chain length of the primer
substrate became longer.
Mutant 2 contained two amino acid
alterations (L34V, R59Q), and L34V was essential for the activity of
GGPP synthesis. This enzyme produced a small amount of GGPP with a
large amount of intermediate products, reflecting the low V value for FPP. The ratio of GGPP/FPP depended
on the allylic substrate used as a primer. The formation of GGPP from
GPP was 10 times as much as that from DMAPP. The K
for DMAPP was increased by this mutation, whereas the K
for GPP was decreased. Moreover, the K
value for IPP in the reaction with GPP as a
primer was the smallest among those in the reaction with the three
allylic substrates, and the V
value for IPP was
the greatest when GPP was used as a primer. These data indicate that
GPP is the best substrate for mutant 2.
Mutant 3 contained two amino acid alterations (V157A, H182Y), and V157A was essential for the GGPP synthase activity. The activity was weaker than those of mutants 1 and 4, and slightly stronger than that of mutant 2. When DMAPP was used, the formation of GPP was quite low, and the ratio of GGPP/FPP, which was 0.24, was similar to that obtained from the reaction using GPP as an allylic substrate.
Mutant 4 contained three amino acid alterations (Y81H, P239R, A265T), and Y81H was essential for amino acid alteration, which was also observed in mutant 1. The pattern of the product distribution was similar to that of mutant 1. However, the kinetic constants slightly differed from those of mutant 1. Mutations of P239R and/or A265T seem to affect the kinetic constants.
By
analyzing kinetic properties of the mutants, we revealed several
factors that might be related to the change from FPP synthase to GGPP
synthase. They include decrease of total reaction rate, increase of K (DMAPP), and change of K
(IPP) depending on an allylic substrate used as a primer.
Detailed mechanisms and kinetic constants of prenyltransferases have
been discussed only for the single condensation of GPP and IPP
catalyzed by avian FPP synthase(33, 34) . The FPP
synthase has been shown to obey the ordered sequential mechanism for
synthesis of FPP from IPP and GPP as shown in . The steady
state kinetic constants can be expressed in terms of individual rate
constant for
Release of FPP is rate-limiting for condensation of IPP and
GPP, where k
50k
, and V
k
[Et]. The slight decrease in V
seen for the mutants using GPP and IPP as
substrates could result from a few hundredfold decrease in the rate of
chemical step (k
), with a concomitant change in
the rate-limiting step, or from an additional slight reduction in
product release step (k
). If the chemical step
becomes rate-limiting, the decrease of k
will
bring about an accumulation of GPP in the reaction between DMAPP and
IPP. However, the mutants did not accumulate GPP (Fig. 2).
Moreover, it is unlikely that the decrease of the chemical step rate
results in the formation of longer chain products. Therefore, the
decrease of V
seems to result from that of k
. If this hypothesis is true, the decrease of V
means the increase of the affinity between FPP
and enzyme. Actually, in mutants 1 and 4, when the reaction was started
with IPP and GPP, the amount of FPP as the intermediate was low. All
mutants showed affinities for FPP to yield GGPP. It is obvious that the
affinity between FPP and the enzyme increases. Moreover, these results
might indicate that the mutation of a prenyltransferase so as to
produce a prenyl product with a longer chain length than that of the
original enzyme is accompanied by sacrifice of the total activity,
which is related to k
.
It has been well known that the prenyltransferases that produce long chain prenyl diphosphates such as hexaprenyl diphosphate, solanesyl diphosphate, and undecaprenyl diphosphate do not accept DMAPP as a priming substrate but need to use GPP, FPP, or GGPP(35, 36, 37, 38) . The mutants show decreased activities for DMAPP. This tendency is prominent in mutants 1 and 4 and is similar to the case of the long chain prenyltransferases described above.
In all mutants, the K values for IPP are dependent on the allylic
substrate that is used as a primer substrate. Especially, in mutant 1,
the K
values for IPP using DMAPP, GPP, and FPP are
265 ± 3.50, 12.4 ± 0.80, and 9.40 ± 1.10
µM, respectively. From the , the increase of K
is brought about not only by the
increase of K
but also by the
decrease of k
. In mutants 1 and 4, there is no
significant difference between the V
values for
DMAPP and GPP. Therefore, it does not seem to be suitable to assume
that k
contributes to the change of K
. If the difference of K
is mainly due to the difference
of K
, this phenomenon indicates
that a single enzyme shows different affinities to the same substrate
depending on countersubstrates. These results might indicate that the
binding of allylic substrate to prenyltransferase causes a
conformational change that affects the affinity of IPP. Moreover,
during the consecutive reaction of prenyltransferase, a series of
conformational changes might occur, and the changes might be essential
for the prenyltransferase reaction. At present, it is unclear which of
the two scenarios is suitable.
We found that the single alteration, Y81H, L34V, or V157A, caused a change of the ultimate product. How are these amino acids involved in catalytic activity? The structural genes for a considerable number of prenyltransferases have been identified and characterized. Comparisons of amino acid sequences of the prenyltransferases were reported by Koyama et al. (14) and Chen et al.(18) , who indicated 7 conserved regions (a-g) and 5 conserved regions, respectively. The conserved regions (a-g), which are proposed by Koyama et al.(14) are indicated in the sequence alignment of chick and B. stearothermophilus FPP synthase (Fig. 3). Leucine at position 34 of B. stearothermophilus FPP synthase is located upstream of region a, which contains the highly conserved GKXXR motif. Tyrosine at position 81 situates in region b, which contains the first DDXXD motif. Prenyltransferases have two conserved DDXX(XX)D aspartate-rich motifs, which are assumed as binding sites for the diphosphate moieties of IPP and the allylic substrates. Valine at position 157 is located in front of region d, which contains the conserved GQXXD motif. However, no significant similarity or difference at the three mutated positions on sequence alignment of prenyltransferases has been observed so far.
Figure 3:
The comparison of the secondary structure
of avian FPP synthase with the predicted secondary structure of B.
stearothermophilus FPP synthase. The comparisons of both the
primary and secondary structures of avian and B. stearothermophilus FPP synthases are shown. In the primary structure, the regions (a, b, c, d, e, f, and g) that have been reported by
Koyama et al. (14) to show significant sequence
conservation are boxed. The essential amino acids, which are
related to chain length determination, are indicated by an underline, and the substituted amino acids are indicated
below. The secondary structure of avian FPP synthase is cited from
Tarshis et al.(41) and indicates above the primary
structure. The letters (A, B, C, D, E, F, G, H, I, J, I,
II,
III) indicated above the secondary structure of avian FPP
synthase are the helix names reported by Tarshis et
al.(41) . The secondary structure drawn below the primary
structure of B. stearothermophilus FPP synthase is essentially
predicted by the methods of Chou and Fasman (39) and Robson (40) .
The secondary structure of B. stearothermophilus FPP
synthase around Tyr-81 was predicted as -helix by the methods of
Chou and Fasman (39) and of Robson (40) (Fig. 3). This secondary structure was also
supported by the crystal structure of avian FPP synthase (Fig. 3)(41) . If the region forms
-helix, the
distance between Tyr-81 and Asp-86, which is the first aspartate of the
first DDXXD motif, is 11
12 Å. The first
DDXXD motif has been thought to bind a diphosphate moiety of
an allylic substrate via magnesium ion. The distance is almost the same
as the length of the hydrocarbon moiety of FPP. These results might
suggest that the side chain of the amino acid blocks further
condensation beyond FPP by direct contact with the hydrocarbon moiety
of FPP. Further analysis of these amino acid residues (Tyr-81, Leu-34,
and Val-157) is necessary to reveal the precise role of the substituted
amino acids and, finally, the mechanism of the chain length
determination.
Recently, Chen et al.(18) have proposed a phylogenetic tree for isoprenyl diphosphate synthases based on a comparative primary structures. In the tree, the most distant separation is between hexaprenyl diphosphate synthase and other synthases. Farnesyl and geranylgeranyl diphosphate synthases segregate into prokaryotic/archaebacterial and eukaryotic families. The separation between FPP synthase and GGPP synthases occurs after the separation between kingdoms. They have postulated that GGPP synthase that produced FPP as an intermediate product is an ancient enzyme based on the phylogenetic tree and the property of archaebacterium Methanobacterium thermoautotrophicum GGPP synthase, which is bifunctional enzyme to produce FPP and GGPP(42) . Our conversion from FPP synthase to GGPP synthase, which also shows the activity of FPP synthase, can be a retrospection of prenyltransferase evolution.