In most types of short-chain all-E prenyl diphosphate synthases, bulky amino acids at the fourth and/or fifth positions upstream from the first aspartate-rich motif play a primary role in
Trang 1type II geranylgeranyl diphosphate synthase requires
subunit interaction
Motoyoshi Noike1,2, Takashi Katagiri1, Toru Nakayama1, Tanetoshi Koyama2, Tokuzo Nishino1and Hisashi Hemmi1
1 Department of Biochemistry and Engineering, Graduate School of Engineering, Tohoku University, Miyagi, Japan
2 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Miyagi, Japan
(All-E) prenyl diphosphate synthase catalyzes the
con-secutive condensation of isopentenyl diphosphates
(IPP) with allylic prenyl diphosphates to yield the final
product with a specific prenyl chain length [1,2] The
chain length of the product must be tightly controlled
because polymerization of isoprene units is the key
reaction responsible for the tremendous variety of naturally occurring isoprenoid compounds (> 50 000) [3] For example, many important compounds, such as carotenoids, tocopherols, diterpenes, chrolophyll and archaeal membrane lipids, are synthesized from gera-nylgeranyl diphosphate (GGPP; C20) On the other
Keywords
farnesyl diphosphate synthase;
geranylgeranyl diphosphate synthase;
isoprenoid; mutagenesis; prenyltransferase
Correspondence
H Hemmi, Department of Applied
Molecular Bioscience, Graduate School of
Bioagricultural Sciences, Nagoya University,
Furo-cho, Chikusa-ku, Nagoya, Aichi
464-8601, Japan
Fax: +81 52 789 4120
Tel: +81 52 789 4134
E-mail: hhemmi@agr.nagoya-u.ac.jp
(Received 21 February 2008, revised 2 June
2008, accepted 4 June 2008)
doi:10.1111/j.1742-4658.2008.06538.x
The product chain length determination mechanism of type II geranyl-geranyl diphosphate synthase from the bacterium, Pantoea ananatis, was studied In most types of short-chain (all-E) prenyl diphosphate synthases, bulky amino acids at the fourth and/or fifth positions upstream from the first aspartate-rich motif play a primary role in the product determination mechanism However, type II geranylgeranyl diphosphate synthase lacks such bulky amino acids at these positions The second position upstream from the G(Q/E) motif has recently been shown to participate in the mechaism of chain length determination in type III geranylgeranyl diphos-phate synthase Amino acid substitutions adjacent to the residues upstream from the first aspartate-rich motif and from the G(Q/E) motif did not affect the chain length of the final product Two amino acid insertion in the first aspartate-rich motif, which is typically found in bacterial enzymes,
is thought to be involved in the product determination mechanism How-ever, deletion mutation of the insertion had no effect on product chain length Thus, based on the structures of homologous enzymes, a new line
of mutants was constructed in which bulky amino acids in the a-helix located at the expected subunit interface were replaced with alanine Two mutants gave products with longer chain lengths, suggesting that type II geranylgeranyl diphosphate synthase utilizes an unexpected mechanism of chain length determination, which requires subunit interaction in the homooligomeric enzyme This possibility is strongly supported by the recently determined crystal structure of plant type II geranylgeranyl diphosphate synthase
Abbreviations
DMAPP, dimethylallyl diphosphate; FARM, first aspartate-rich motif; FPP, farnesyl diphosphate; FPS, farnesyl diphosphate synthase; GGPP, geranylgeranyl diphosphate; GGPS, geranylgeranyl diphosphate synthase; GPP, geranyl diphosphate; GPS, geranyl diphosphate synthase; IPP, isopentenyl diphosphate; SARM, second aspartate-rich motif.
Trang 2hand, farnesyl diphosphate (FPP; C15) is the precursor
of steroids, sesquiterpenes and heme a Moreover, FPP
is the usual allyic primer substrate for prenyl
elonga-tion reacelonga-tions, which yield longer-chain prenyl
diphos-phates as the precursors of respiratory quinones,
dolichol and natural rubber, although some organisms
also use GGPP for the same purpose Longer-chain
(all-E) prenyl diphosphates (up to C60) are utilized for
the biosynthesis of various respiratory quinones, which
have been used to classify the microorganisms GGPP
and FPP are also utilized for protein modification,
although they modify very different classes of acceptor
proteins Rab family G proteins are
geranylgeranyl-ated, whereas farnesylation typically occurs on Ras
proteins In addition, geranyl diphosphate (GPP; C10)
is the precursor of volatile monoterpenes and also is
used to modify secondary metabolites
The mechanism of prenyl-chain elongation, and
therefore of product determination in (all-E) prenyl
diphosphate synthases, which share many conserved
sequences in spite of their different reaction products,
has been investigated previously The enzymes are
con-structed mainly of a-helices, which form a large
reac-tion cavity per a subunit [4–12] Most of the enzymes
are homodimeric proteins, although some enzymes
consist of heterodimers with little homology between
the subunits [1] A few mammalian enzymes are known
to form oligomers [13,14] The highly conserved motifs
of the enzymes [i.e the first aspartate-rich motif
(FARM) and the second aspartate-rich motif (SARM)]
are thought to bind the diphosphate group of the allylic
substrate via magnesium ions FARM and SARM are
located on a-helices D and H (Note that the present
study follows the helix designation first reported for the
crystal structure of avian farnesyl diphosphate synthase
(FPS) by Tarsis et al [4].) Departure of the
diphos-phate group forms an allylic carbocation, which is
attacked by the p-electron at the double-bond of IPP,
forming a new bond between the fourth carbon of IPP
and the first carbon of the allylic substrate Thus,
pre-nyl diphosphate is elongated by one C5 prenyl unit
The condensation reaction is repeated, elongating the
prenyl chain As the chain elongates, the hydrocarbon
moiety becomes located deep within the reaction cavity
formed by a-helices C, D, E, F, G and H
Enzyme-specific termination of prenyl-chain elongation results
in final products unique to each enzyme
Mutational and structural studies have revealed that,
in general, bulky amino acids at the bottom of the
cav-ity block prenyl-elongation In particular, our research
group has shown that, in (all-E) prenyl diphosphate
synthases yielding short-chain products such as GGPP
and FPP, the bulky amino acids are found in two
regions: upstream from FARM [15] and from the highly-conserved G(Q/E) motif [16], respectively FARM exists on a-helix D and the G(Q/E) motif is located on a-helix F Based on the characteristic sequences upstream from FARM, the short-chain enzymes have been classified into five types [15]: three types of geranylgeranyl diphosphate synthase (GGPS) and two types of FPS (Fig 1) Type I GGPS from archaea has a bulky aromatic amino acid residue, which plays a primary role in the chain length determi-nation mechanism at the fifth position upstream from FARM The importance of the residue was shown by mutational studies on GGPS from a thermoacidophilic archaeon Sulfolobus acidocaldarius [17–19] Two GGPSs with known crystal structures [i.e those from
a hyperthermophilic archaeon Pyrococcus horikoshii (Protein Data Bank code 1WY0) and from a thermo-philic bacterium Thermus thermophilus (1WMW)] also fall into this type The bulky residue at the fifth posi-tion upstream from FARM is in the center of the cavity and likely to act as the bottom of it in these structures; however, the structural information is inde-cisive with respect to the role of the residue because the structures do not contain allylic substrates or their analogues bound in the active site Such characteristic
Fig 1 Alignment of amino acid sequences around FARM of vari-ous (all-E) prenyl diphosphate synthases The partial amino acid sequences of the enzymes classified into two types of FPSs synth-ases and three types of GGPSs are aligned Sce FPS, S cerevisiae FPS; Gga FPS, Gallus gallus (avian) FPS; Eco FPS, E coli FPS; Gst FPS, G stearothermophilus FPS; Sac GGPS, S acidocaldarius GGPS; Mth GGPS, Methanobacterium thermoautotrophicum GGPS; Pan GGPS, P ananatis GGPS; Sal GGPS, S alba (mustard) GGPS; SceGGPS, S cerevisiae GGPS; Hsa GGPS, Homo sapiens GGPS The characteristic amino acid residues suggested to be involved product determination for each type of enzyme are shaded.
Trang 3sequences also are evident in FPSs Eukaryotic type I
FPS has bulky amino acids at both the fourth and fifth
positions upstream from FARM Mutational and
structural studies on avian FPS clarified the role of the
positions [20] In the structure of mutated avian FPS
(1UBX), in which phenylalanine residues at the fourth
and fifth positions upstream from FARM are replaced
with serine and alanine, respectively, the x-end of the
hydrocarbon chain of FPP bound in the cavity passes
through the hole formed by the mutagenesis Broad
structural studies using inhibitor substrate analogues
were made with type 1 FPSs from human [11] and
pro-tozoa [6,12], and some of the structures [e.g FPS from
human (2F94 and 3B7L) and Trypanosoma brucei
(2P1C and 2I19) binding bisphosphonate inhibitors
with hydrocarbon chains as long as that of GPP]
sug-gest the importance of the bulky amino acids because
they are in contact with the inhibitors However, no
mutational study that supports the hypothesis has been
made with the enzymes Bacterial type II FPS has a
bulky amino acid only at the fifth upstream position,
but also has a two amino acid insertion in FARM
Mutational studies of FPS from Geobacillus
stearother-mophilus showed that the bulky amino acid upstream
from FARM is involved in the chain length
determina-tion mechanism [21,22] The crystal structure of this
type of FPS was elucidated using the enzymes from
several bacteria, such as Staphylococcus aureus and
Escherichia coli[7] The structures of E coli FPS
bind-ing substrate analogues (1RQI and 1RQJ) also suggest,
but do not ensure, the role of tyrosine at the fifth
posi-tion upstream from FARM, which is still distant from
the analogues with short hydrocarbon chains used in
that study [7] By contrast, eukaryotic type III GGPS,
which lacks bulky amino acids at the fourth or fifth
positions upstream from FARM, was shown to utilize
bulky amino acids at the second position upstream
from the G(Q/E) motif to terminate chain elongation
by our mutational work using GGPS from
Saccharo-myces cerevisiae [16] This information was later
supported by a structural and mutational study on the
same enzyme (2DH4) [9]
In the present study, mutational studies of GGPS
from a bacterial plant pathogen, P ananatis, were
performed to investigate the mechanism of chain
length determination in type II GGPS from bacteria
and plants, which has not been identified to date
This type of GGPS lacks bulky aromatic amino acids
at the fourth and fifth positions upstream from
FARM, similar to type III GGPS, whereas it has a
two amino acid insertion in FARM, as does type II
FPS Unexpectedly, mutations at the fourth and fifth
positions upstream from FARM and at the second
position upstream from the G(Q/E) motif did not affect the chain length of the final product In addi-tion, deletion of the insertion sequence in FARM, which is thought to be involved in the chain length determination mechanism [18], also had no effect on the chain length of the final product An additional mutational study with type II FPS from G stearother-mophilus confirmed that the insertion in FARM does not play a role in the mechanism of chain length determination in type II enzymes These results sug-gest that chain length determination is controlled by another region of the enzymes Thus, a new line of mutants was created based on the crystal structures
of other short-chain enzymes and on the results from previous mutational studies Accordingly, a-helix E, which would be located at the subunit interface of the enzyme, was identified as playing a role in the product chain length determination mechanism of type II GGPS Moreover, this result suggests that the other subunit of the homooligomeric enzyme is involved in the product chain length determination mechanism This conclusion is supported by the recently-solved crystal structure of type II GGPS from mustard [23] The mechanism of product chain length determination of type II GGPS identified in the present study may also explain the participation
of noncatalytic subunits in the product determination mechanisms of some heteromeric enzymes, such as geranyl diphosphate synthase (GPS) and longer-chain prenyl diphosphate synthases
Results
Refolding and purification of recombinant
P ananatis GGPS
P ananatis GGPS and the mutant enzymes were expressed in E coli as inclusion bodies To obtain sol-uble enzymes, inclusion bodies prepared from the insoluble fraction were denatured by guanidine hydro-chloride and then purified by refolding on a HisTrap column The purified proteins gave almost single, iden-tical bands by SDS/PAGE (data not shown) Only the mutant L128A was completely inactive All other mutant GGPSs exhibited enzyme activity comparable
to that of the wild-type enzyme, whereas L127A showed only approximately 20% activity of wild-type Analysis of the quaternary structure of the refolded enzyme using blue native PAGE showed that the molecular mass of P ananatis GGPS is approximately
130 or 240 kDa, suggesting that the main part of the enzyme exists as a homotetramer or a homooctamer (Fig 2)
Trang 4Mutation in the region upstream from FARM
In type II GGPS, bulky aromatic amino acids at the
fourth and/or fifth positions upstream from FARM,
which are characteristic of many short-type (all-E)
pre-nyl diphosphate synthases, are not present Previous
studies, mainly conducted by our research group,
indi-cated that bulky amino acids on a-helix D, which
includes FARM, block prenyl-chain elongation,
thereby controlling chain length [17–20,22] To
mine whether this mechanism of chain length
deter-mination also operates in type II GGPS from
P ananatis, alanine 89 at the fifth position upstream
from FARM was replaced with bulky amino acids
(Fig 3A) These mutations were designed to mimic
bacterial type II FPS The mutants, A89F, A89L and
A89H, yielded shorter products than the wild-type
enzyme (Fig 3B) In particular, the substrate
specifici-ties of A89F and A89H were almost identical to that
of FPS: product yield was minimal when GGPP was
used as the substrate This result suggested that, in
type II GGPS, the prenyl-chain of the product
elon-gates along a-helix D and that the amino acid residues
on a-helix D further upstream from FARM are
involved in chain length determination Thus, new
mutations were introduced further upstream from
FARM It was expected that substituting the smaller
amino acid, alanine, for the bulky residues would
increase the chain length of the final products
(Fig 4A) However, these mutants (i.e., H87A, V86A
and M85A) did not yield longer products than the
wild-type (Fig 4B) H87A activity using GGPP as the
substrate was undetectable, probably because the
mutation significantly decreased overall enzyme
activ-ity These results clearly indicated that bulky amino
acids relative to FARM do not contribute to the prod-uct determination mechanism of type II GGPS
Mutation at the second position upstream from the G(Q/E) motif
Because the mechanism of chain length determination for type II GGPS was shown to be independent from the region upstream from FARM, the other region known to play a role in chain length determination was expected to play a critical role The second posi-tion upstream from the conserved G(Q/E) motif was first identified as an important residue in the chain length determination mechanism of type III GGPS from S cerevisiae in a previous study conducted in our laboratory [16] The relatively bulky residue, histidine
139, rather than those upstream from FARM, was found to block chain-elongation The role of the resi-due was later supported by Chang et al [9]: the crystal structure of S cerevisiae GGPS that these authors determined demonstrated that histidine 139 forms the bottom of the reaction cavity Therefore, mutants of type II GGPS from P ananatis, in which the residue
at the second position upstream from the G(Q/E)
Fig 2 Blue native PAGE of refolded P ananatis GGPS The
refold-ing procedure is described in the Experimental procedures Lane 1,
molecular mass standard; lane 2, wild-type; lane 3, I121A; lane 4,
V125A.
A
B
Fig 3 Introduction of substitutive mutations into the fifth position upstream from FARM of P ananatis GGPS (A) Partial amino acid sequences around FARM of wild-type and mutated enzymes are aligned The substituted amino acid residues are shaded (B) TLC autoradiochromatograms of the reaction products of wild-type and mutated enzymes The products were analyzed as described in the Experimental procedures The allylic substrate used is indicated at the top of each autoradiochromatogram Lane 1, A89F; lane 2, A89L; lane 3, A89H; lane 4, wild-type Under all assay conditions,
< 30% of each substrate reacted Ori., origin; S.F., solvent front.
Trang 5motif was replaced with smaller amino acids, were
con-structed (Fig 5A) However, the mutants, V163A,
V163G and V163S, did not yield products with a chain
length longer than those produced by the wild-type
enzyme (Fig 5B) Moreover, some mutants did not
give the C25side-product and exhibited decreased
spec-ificity for GGPP This unexpected result indicated that
the second position upstream from the G(Q/E) motif
does not contribute to the mechanism of chain length
determination in type II GGPS In addition,
substi-tution of V163 with a bulky amino acid,
phenylala-nine, resulted in loss of activity (data not shown)
Deletion of the insertion sequence in FARM
Ohnuma et al [18] performed a detailed investigation
of the mechanism of chain length determination in
short-chain (all-E) prenyl diphosphate synthase, mainly
using type I GGPS from S acidocaldarius to construct
various mutants In their study, two amino acids were
inserted in FARM of type I GGPS to mimic type II
FPS because this two amino acid insertion, which is
specifically observed in type II FPS and type II GGPS,
was expected to affect the product chain length Type
I GGPS with the insertion mutation yielded larger
amounts of the reaction intermediate, FPP, whereas GGPP remained the final product Although Ohnuma
et al [18] did not confirm the effect of the insertion by performing the converse mutation (i.e deletion of the insertion from type I FPS or type II GGPS), the inser-tion sequence was thought to play a role in the mecha-nism of chain length determination in type II GGPS Thus, in the present study, the two amino acids inser-tion was deleted from FARM of type II GGPS from
P ananatis to confirm the effect of the deletion on product chain length (Fig 6A) However, the mutant, GGPS-DFARM, did not yield a final product with a chain length longer than that of the product resulting from the wild-type enzyme, which gave a small amount
of the C25 side-product (Fig 6C) The mutant enzyme appeared to exhibit reduced activity toward GGPP, although this reduction may have been due to a decrease in overall enzyme activity This result indi-cates that the insertion does not have a significant effect on the mechanism of chain length determination
in type II GGPS In addition, the two amino acid insertion in FARM was deleted from type II FPS
of G stearothermophilus (Fig 6B) The mutant FPS-DFARM also showed product specificity similar
A
B
Fig 4 Introduction of substitution mutations into the region
upstream from FARM of P ananatis GGPS (A) Partial amino acid
sequences around FARM of wild-type and mutated enzymes are
aligned The substituted amino acid residues are shaded (B) TLC
autoradiochromatograms of the reaction products of wild-type and
mutated enzymes The products were analyzed as described in the
Experimental procedures The allylic substrate used is indicated at
the top of each autoradiochromatogram Lane 1, H87A; lane 2,
V86A; lane 3, M85A; lane 4, wild-type Under all assay conditions,
< 30% of each substrate reacted Ori., origin; S.F., solvent front.
A
B
Fig 5 Introduction of substitution mutations into the second posi-tion upstream from the G(Q/E) motif of P ananatis GGPS (A) Par-tial amino acid sequences around the G(Q/E) motif of wild-type and mutated enzymes are aligned The substituted amino acid residues are shaded (B) TLC autoradiochromatograms of the reaction prod-ucts of the wild-type and mutated enzymes The prodprod-ucts were analyzed as described in the Experimental procedures The allylic substrate used is indicated at the top of each autoradiochromato-gram Lane 1, V163A; lane 2, V163G; lane 3, V163S; lane 4, wild-type Under all assay conditions, < 30% of each substrate reacted Ori., origin; S.F., solvent front.
Trang 6to that of the wild-type FPS, although the
characteris-tics around FARM mimicked those of type I GGPS
(Fig 6D) Therefore, it was concluded that the two
amino acid insertion does not play an important role
in the chain length determination mechanism in either
type II GGPS or type II FPS
Mutation in a-helix E
In the mutational study on type III GGPS from S
ce-revisiae conducted by Chang et al [9], the bottom of
the reaction cavity was suggested to be comprised not
only of histidine 139 at the second position upstream
from the G(Q/E) motif, but also of tyrosine 107 and
phenylalanine 108 Substituting alanine for tyrosine
107 and phenylalanine 108 increased the chain length
of the final products, as did histidine 139 These bulky
residues exist in proximity in the structure of the
enzyme, which was also reported in the same study
Tyrosine 107 and phenylalanine 108 are located in
a-helix E (Chang et al [9] referred to a-helix E as
a-helix F), whereas FARM and the G(Q/E) motif are located in a-helices D and F, respectively (D and G according to the designation of Chang et al [9]) Moreover, the structure of S cerevisiae GGPS binding GGPP recently reported (2E8V) revealed that tyrosine
107 directly touches the x-end of GGPP bound in the same subunit, whereas phenylalanine 108 supplied from the other subunit exists in the proximity of the x-end [24]
These results led to the hypothesis that the prenyl-chain of the product elongates in the space enclosed by a-helices D, E and F, and that the bulky amino acid residues on at least one of the a-helices block chain-elongation If this hypothesis is correct, type II GGPS should use residues on a-helix E to terminate chain-elongation Thus, alanine substitution mutations were introduced at each position on a-helix E where a bulky amino acid was located (Fig 7A) These bulky amino acids on a-helix E can form the bottom of a reaction cavity similar to those residues located at the key posi-tions upstream from FARM and from the G(Q/E)
A
B
Fig 6 Deletion of the insertion sequences in FARM of P ananatis GGPS and G stearothermophilus FPS (A) Partial amino acid sequences around FARM of P ananatis GGPS and mutated enzymes are aligned The deleted positions are shaded (B) Partial amino acid sequences around FARM of G stearothermophilus FPS and mutated enzymes are aligned The deleted positions are shaded (C) TLC autoradiochroma-tograms of the reaction products of the P ananatis GGPS and mutated enzymes The products were analyzed as described in the Experi-mental procedures The allylic substrate used is indicated at the top of each autoradiochromatogram Lane 1, GGPS-DFARM; lane 2, wild-type GGPS Under all assay conditions, < 30% of each substrate reacted Ori., origin; S.F., solvent front (D) TLC autoradiochromato-grams of the reaction products of the G stearothermophilus FPS and mutated enzymes The products were analyzed as described in the Experimental procedures The allylic substrate used is indicated at the top of each autoradiochromatogram Lane 1, FPS-DFARM; lane 2, wild-type FPS Under all assay conditions, < 30% of each substrate reacted Ori., origin; S.F., solvent front.
Trang 7motif in the other types of the enzyme Among the
constructed mutants, I121A and V125A yielded longer
products than the wild-type enzyme (Fig 7B,C) I121A
gave a C35product when GGPP was used as the
sub-strate, whereas the final product of the wild-type
GGPS was C25 prenyl diphosphate On the other
hand, V125A yielded a series of products whose maxi-mum chain length reached over C40 when GPP or GGPP was used as the primer substrate The other mutants showed product specificity similar to that of the wild-type enzyme, although some mutants exhib-ited negligible substrate specificity for GGPP
A
B
C
Fig 7 Introduction of substitutive mutations into the predicted a-helix E of P ananatis GGPS (A) Partial amino acid sequences around a-helix E of wild-type and mutated enzymes are aligned The substituted amino acid residues are shaded (B) TLC autoradiochromatograms
of the reaction products of wild-type and mutated enzymes The products were analyzed as described in the Experimental procedures The allylic substrate used is indicated at the top of each autoradiochromatogram Lane 1, L122A; lane 2, I121A; lane 3, H118A; lane 4, E117A; Lane 5, Y115A; lane 6, H114A; lane 7, wild-type Under all assay conditions, < 30% of each substrate reacted Ori., origin; S.F., solvent front (C) TLC autoradiochromatograms of the reaction products of wild-type and mutated enzymes Lane 1, V125A; lane 2, L127A; lane 3, wild-type.
Trang 8In the present study, type II GGPS from P ananatis
was recombinantly expressed and purified by refolding
on a column Blue native PAGE suggested that the
enzyme has a homotetrameric or homooctermeric
structure Crystal structural analysis of human GGPS
reveals that three homodimers, which comprise the
same quaternary structure observed for most
short-chain (all-E) prenyl diphosphate synthases, join
together to form homohexamer [9] Thus, in the case
of P ananatis GGPS, it also is likely that two or four
homodimers join together to form a homotetramer or
a homooctamer, respectively The recombinant enzyme
and its mutants were used to identify amino acid
resi-dues that contribute to the mechanism of chain length
determination Unexpectedly, the two regions that are
known to play important roles in the mechanism in
some types of short-chain (all-E) prenyl diphosphate
synthases [i.e the fourth and fifth positions upstream
from FARM and the second position upstream from
the G(Q/E) motif] were not involved in chain length
determination in type II GGPS Moreover, a two
amino acid insertion in FARM, which was thought to
be involved in the mechanism of chain length
determi-nation, had no significant effect on the product chain
length in either type II GGPS or type II FPS
Alterna-tively, alanine substitution mutations in a-helix E
revealed that isoleucine 121 and valine 125 are the
resi-dues involved in the mechanism of chain length
deter-mination To the best of our knowledge, this is the
first report to describe mutations in type II GGPS that
change the chain length of the final product of the
enzyme
Although it was apparent that a-helix E was
involved in the mechanism of chain length
determina-tion in type II GGPS, an addidetermina-tional quesdetermina-tion was
raised The crystal structures of some of the
homodi-meric (all-E) prenyl diphosphate synthases indicated
that a-helix E exists at the dimer interface Thus, the
question arose as to whether the critical residues (i.e
I121 and V125) provided for the reaction cavity are
from the same catalytic subunit or from the other
pair-ing subunit? Fortunately, a crystal structure that was
recently solved has provided a clear answer to this
question Kloer et al [23] reported the crystal structure
of type II GGPS from mustard (Sinapis alba), binding
GGPP In the homodimeric structure (2J1P), the
gera-nylgeranyl chain of GGPP elongates in the cavity
formed by four a-helices, D, E, F (from the catalytic
subunit) and E¢ (from the pairing subunit) V178¢ and
D182¢ in a-helix E¢, which correspond to I121 and
V125 of P ananatis GGPS, respectively, exist much
closer to the geranylgeranyl chain than do V178 and D182 in a-helix E (Fig 8A) Especially, D182¢ directly touches the x-end of the geranylgeranyl-chain Although V178¢ is not in direct contact with GGPP, it appears to support D182¢ or L179¢ at the next position
in a-helix E¢, which touches the center of the geranyl-geranyl-chain and bends it towards the bottom of the cavity formed by L185 in a-helix E, I216 in a-helix F, and D182¢ and S186¢ in a-helix E¢ (Fig 8B, left) The fourth and fifth residues upstream from FARM [i.e S147 and MSE (selenomethionine)146, respectively] also are in contact with the geranylgeranyl chain, but these residues appear to act only as part of the cavity wall, as does the second residue upstream from the
GQ motif (i.e V222) An almost similar spatial arrangement was observed in the model structure of
A
B
Fig 8 Structural information on the product determination mecha-nism of type II GGPS (A) The direction of the geranylgeranyl chain
of GGPP bound in a subunit (blue) of S alba GGPS The x-end of the chain touches a-helix E¢ supplied from the other subunit (pink) GGPP is indicated by a cylinder model and some equivalent resi-dues on a-helices E and E¢ are shown as sphere models (B) Close view of the substrate pocket of S alba GGPS (left) and that of the modeled dimeric structure of P ananatis GGPS (right) The model
of P ananatis GGPS was constructed based on the crystal struc-ture of S alba GGPS as the template Some of the amino acid residues surrounding the geranylgeranyl chain of GGPP bound in
S alba GGPS and the corresponding residues in P ananatis GGPS are indicated as sphere models The geranylgeranyl chain is indicated by green cylinders.
Trang 9P ananatis GGPS, which was constructed using the
crystal structure of S alba GGPS as the template for
molecular modeling (Fig 8B, right) From this
struc-tural information, it is readily apparent that
replace-ment of V178 or D182 of S alba GGPS with a smaller
amino acid would create a new chain-elongation path
through which the prenyl chain can lengthen across
the subunit interface It is conceivable that a similar
scenario also occurred in the I121A and V125A
mutants of P ananatis GGPS
The role of a-helix E in the mechanism of chain length
determination also has been reported for a few
short-chain (all-E) prenyl diphosphate synthases of other
types As mentioned above, type III GGPS from S
cere-visiae utilizes tyrosine 107 and phenylalanine 108 to
terminate elongation of the product [9], although these
residues do not correspond with the positions 121 and
125 of P ananatis GGPS The recently reported
struc-ture of the complex of S cerevisiae GGPS and GGPP
revealed that tyrosine 107 was supplied from the other
subunit [24], as is the case for I121 and V125 in type II
GGPS from P ananatis On the other hand, Lee et al
[25] reported that, in type II FPS from E coli, glycine
substitution of aspartate 115, which also exists in a-helix
E, allows the enzyme to produce GGPP The position of
the aspartate residue corresponds to V125 in P ananatis
GGPS Lee et al [25] hypothesized that the destruction
of the hydrogen bond between aspartate 115 and
histi-dine 83 increases the flexibility of the a-helices,
expand-ing the reaction cavity In their paper it is likely that
only the arrangement of the residues in a monomer
sub-unit was considered as an explanation of the
phenome-non However, in the crystal structure of E coli FPS
binding IPP and the analogue of dimethylallyl
diphos-phate (DMAPP) (1RQI) [7], aspartate 115 exists in close
proximity to the tyrosine 79¢ at the fifth position
upstream from FARM of the other subunit, which is
considered to play a significant role in chain length
determination This observation strongly suggests that
the aspartate residue might be supplied to offer
struc-tural support of tyrosine 79¢ or to block
chain-elonga-tion, probably in part, in the other subunit
The results of the present study, which suggest the
requirement of subunit interaction for chain length
determination in type II GGPS, are reminiscent of an
intriguing study by Burke and Croteau [26] These
authors reported that a subunit of homodimeric type
II GGPS from Taxus candensis and a small subunit
of heterotetrameric GPS from Mentha piperita can
form a hybrid heterodimer, which yields GPP when
DMAPP is used as the substrate The large subunit of
M piperitaGPS is very similar to T candensis GGPS,
whereas the small subunit is not Thus, the small,
probably noncatalytic subunit was shown to influence the product specificity of type II GGPS It is conceiv-able that the mechanism that acts in P ananatis GGPS
is similar to that observed for the hybrid heteromeric enzyme and to the mechanism that likely occurs in het-eromeric GPSs from plants Hethet-eromeric longer-chain (all-E) prenyl diphosphate synthases have been identi-fied, including heptaprenyl diphosphate (C35) synthases from bacilli [27–29]; hexaprenyl diphosphate (C30) syn-thase from Micrococcus luteus B-P 26 [30]; solanesyl diphosphate (C45) synthase from mouse [31]; and deca-prenyl diphosphate (C50) synthase from human [31] and Schizosaccharomyces pombe [32] These
heteromer-ic enzymes may provide more definitive evidence for subunit interaction in the mechanism of chain length determination Indeed, Zhang et al [33] reported that mutation in the small subunit of heterodimeric hepta-prenyl diphsophate synthase from Bacillus subtilis, which shows only slight similarity with homodimeric (all-E) prenyl diphosphate synthases, affects the chain length of the final product
As noted above, the crystal structures of avian FPS and human GGPS as the complexes with their final products, FPP and GGPP, respectively, have been solved [10,20] However, the direction of prenyl-chain elongation differs between these enzymes In the struc-ture of mutated avian type I FPS binding FPP (1UBX), reported as the monomeric form, the farnesyl chain elongates toward the expected dimer interface [20] Thus, the cavity of avian type I FPS is thought to
be constructed by a-helices D, E, F and probably E¢,
as is that of type II GGPS from S alba By contrast,
in human GGPS binding GGPP (2Q80), the x-end of the geranylgeranyl chain enters the space enclosed by a-helices C, D and G [10] In the structure, the resi-dues known to be important in the chain length deter-mination [i.e the fourth and fifth positions upstream from FARM and the second position upstream from the G(Q/E) motif] just come into contact with the product at the center of the prenyl chain, suggesting that these residues do not act to form the bottom of the cavity in human type III GGPS A similar path of prenyl-chain elongation was suggested for hexaprenyl diphosphate synthase from Sulfolobus solfataricus In a mutational study, alanine or glycine substitution for leucine 164 in a-helix G increased the chain length of the final product [8] However, enzymes with chain-elongation paths enclosed by a-helices C, D and G might be exceptional because the structural studies of the other (all-E) prenyl diphosphate synthases, includ-ing type III GGPS from S cerevisiae [24] and octapre-nyl diphosphate synthase from Thermotoga maritima [34], as well as a large number of mutational studies,
Trang 10suggest that the majority of enzymes have paths
enclosed by a-helices D, E and F In the enzymes
pos-sessing structures similar to human GGPS, it is
possi-ble that a different type of chain length determination
mechanism exists in which amino acid residues in
unknown regions play crucial roles
Experimental procedures
Materials
Precoated reversed-phase TLC plates, LKC-18F were
pur-chased from Whatman (Maidstone, UK) (all-E) GGPP,
(all-E) FPP and (all-E) GPP were synthesized as previously
reported [35] Nonlabeled IPP and DMAPP were donated
by C Ohto (Toyota Motor Co., Japan) [1-14C]IPP was
purchased from GE Healthcare (Piscataway, NJ, USA) All
other chemicals were of analytical grade
General procedures
Restriction enzyme digestions, transformations and other
standard molecular biology techniques were carried out as
previously described [36]
Plasmid construction and site-directed
mutagenesis
Using pACYC-IBE [37], which contains carotenoid
biosyn-thetic genes from P ananatis, as the template, the crtE gene
encoding GGPS was amplified using PCR with KOD DNA
polymerase (Toyobo, Osaka, Japan) and the primers: PaG
GPS-Fw, 5¢-AAGAAACATATGACGGTCTGCGCAAA
AAAACACG-3¢, and PaGGPS-Rv, 5¢-TGCAGAGGATCC
TTAACTGACGGCAGCGAGTTTTTTG-3¢ The
sequen-ces corresponding to the NdeI and BamHI sites that were
used in subsequent experiments are underlined in the
pri-mer sequences above The amplified fragment was cleaved
with the restriction enzymes and then inserted into an
NdeI/BamHI-treated pET-15b vector (Novagen, Madison,
WI, USA) to construct pET-HisPaGGPS, a plasmid for the
recombinant expression of His6-tagged P ananatis GGPS
For the expression of His6-tagged G stearothermophilus
FPS, the gene was amplified using PCR with KOD DNA
polymerase (Toyobo), a pFPS [21], and the primers:
His-BsFPS-Fw, 5¢-ACAGCCATGGGACATCATCATCATCA
TCATGCGCAGCTTTCAGTTGAA-3¢, and
HisBsFPS-Rv, 5¢-TGAATTTAAAGCTTAATGGTCGCGGGCG-3¢
The sequences corresponding to the NcoI and HindIII sites
that were used in subsequent experiments are underlined in
the above sequences The amplified fragment was cleaved
with the restriction enzymes and then inserted into the
NcoI/HindIII-treated pTV118N vector (TaKaRa, Shiga,
Japan) to construct pTV-HisBsFPS Site-directed mutations
were introduced into each parental plasmid utilizing a QuikChange Mutagenesis Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s instructions
Expression and purification of wild-type and mutated enzymes
For the expression of P ananatis GGPS, E coli BL21(DE3) was transformed with pET-HisPaGGPS or the mutated plasmids The transformants were cultivated in
50 mL of M9 minimal broth supplemented with glycerol (2 gÆL)1), yeast extract (2 gÆL)1) and ampicillin (50 mgÆL)1) When D600of 0.5 was reached, the transformed bacteria in the culture were induced with 1.0 mm isopropyl thio-ß-d-galactoside The cells were incubated overnight and then harvested The cells were disrupted in lysis buffer contain-ing 20 mm sodium phosphate buffer (pH 8.0), 10 mm imidazol and 0.5 m NaCl The homogenate was centrifuged
at 6000 g for 15 min at 4C and the precipitate containing the inclusion body enzyme was recovered The precipitate was dissolved in lysis buffer supplemented with 4% Triton X-100 After shaking for 30 min at 25C, the mixture was centrifuged at 6000 g for 15 min at 4C and the precipitate was recovered The process was repeated twice to remove bacterial membranous compounds The washed precipitate was lyophilized and used as the inclusion body Ten milligrams of the inclusion body was dissolved in
10 mL of denaturation buffer containing 50 mm sodium phosphate buffer (pH 8.0), 10 mm imidazol, 6 m guanidine hydrochloride, 10 mm dithiothreitol, 10 mm 2-mercapto-ethanol and 0.5 m NaCl After centrifugation at 9000 g for
15 min at 4C, the supernatant was recovered and then applied to a HisTrap column (GE Healthcare) equilibrated with equilibration buffer containing 50 mm sodium phosphate buffer (pH 8.0), 10 mm imidazol, 6 m guanidine hydrochloride, 10 mm dithiothreitol, 10 mm 2-mercapto-ethanol and 0.5 m NaCl The column was washed with
10 mL of equilibration buffer and then with start buffer con-taining 50 mm sodium phosphate buffer (pH 8.0), 10 mm im-idazol, 10 mm 2-mercaptoethanol and 0.5 m NaCl to remove the denaturant The protein renatured in the column was eluted from the column with 10 mL of elution buffer contain-ing 50 mm sodium phosphate buffer (pH 8.0), 500 mm imi-dazol, 10 mm 2-mercaptoethanol and 0.5 m NaCl The eluate was fractioned, and the fraction with the highest enzyme activity and purity was used as the partially purified enzyme
in the experiments described below The purity of the enzyme was determined by 15% SDS/PAGE
For the expression of G stearothermophilus FPS, E coli DH5a was used as the host The transformants were culti-vated and induced as described above Disruption of harvested cells and the purification of the tagged enzymes were conducted utilizing a MagExtractor His-tag Kit (Toyobo) according to the manufacturer’s instructions