In contrast, mechanisms for determination of the ultimate product chain length of cis-prenyltransferases have not yet been determined although mutational analysis of highly conserved res
Trang 1mechanism of cis-prenyltransferases
Yugesh Kharel*, Seiji Takahashi*, Satoshi Yamashita and Tanetoshi Koyama
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan
In the biosynthesis of isoprenoids, which are the most
structurally diverse and abundant among natural
prod-ucts, all carbon skeletons are biosynthesized using
sequential condensation of isopentenyl diphosphate
(IPP, C5) and allylic diphosphates by actions of prenyl
chain elongating enzymes, commonly called
prenyl-transferases Prenyltransferases can be classified into
two major groups, i.e trans- and cis-prenyltransferases,
according to the geometry of the prenyl chain units in
the products [1,2] The reactions catalysed by
prenyl-transferases start by the formation of allylic cation
after the elimination of pyrophosphate ion to form an allylic prenyl diphosphate, followed by addition of an IPP with stereospecific removal of a proton at the 2-position The only difference between the reaction catalyzed by trans- and cis-prenyltransferase is the prochirality of the proton, i.e pro-R for trans-prenyltransferase and pro-S for cis-trans-prenyltransferase (Fig 1a) However, recent molecular analysis and crys-tal structure determination of Micrococcus luteus B-P
26 undecaprenyl diphosphate (UPP, C55) synthase, which catalyses cis-condensation to synthesize UPP,
Keywords
chain-length determination mechanism;
cis-prenyltransferase; isoprenoid biosynthesis;
Micrococcus luteus B-P 26; undecaprenyl
diphosphate synthase
Correspondence
T Koyama, Institute of Multidisciplinary
Research for Advanced Materials, Tohoku
University, Katahira 2-1-1, Aoba-ku, Sendai,
980-8577, Japan
Fax: +81 22 217 5620
Tel:: +81 22 217 5621
E.mail: koyama@tagen.tohoku.ac.jp
Note
*Both authors contributed equally to this
work.
(Received 28 October 2005, revised 9
December 2005, accepted 12 December
2005)
doi:10.1111/j.1742-4658.2005.05097.x
The carbon backbones of Z,E-mixed isoprenoids are synthesized by sequential cis-condensation of isopentenyl diphosphate (IPP) and an allylic diphosphate through actions of a series of enzymes called cis-prenyltrans-ferases Recent molecular analyses of Micrococcus luteus B-P 26 undecapre-nyl diphosphate (UPP, C55) synthase [Fujihashi M, Zhang Y-W, Higuchi
Y, Li X-Y, Koyama T & Miki K (2001) Proc Natl Acad Sci USA 98, 4337–4342.] showed that not only the primary structure but also the crystal structure of cis-prenyltransferases were totally different from those of trans-prenyltransferases Although many studies on structure–function rela-tionships of cis-prenyltransferases have been reported, regulation mecha-nisms for the ultimate prenyl chain length have not yet been elucidated
We report here that the ultimate chain length of prenyl products can be controlled through structural manipulation of UPP synthase of M luteus B-P 26, based on comparisons between structures of various cis-prenyl-transferases Replacements of Ala72, Phe73, and Trp78, which are located
in the proximity of the substrate binding site, with Leu) as in Z,E-farnesyl diphosphate (C15) synthase) resulted in shorter ultimate products with
C20)35 Additional mutation of F223H resulted in even shorter products
On the other hand, insertion of charged residues originating from long-chain cis-prenyltransferases into helix-3, which participates in constitution
of the large hydrophobic cleft, resulted in lengthening of the ultimate prod-uct chain length, leading to C60)75 These results helped us understand reaction mechanisms of cis-prenyltransferase including regulation of the ultimate prenyl chain-length
Abbreviations
DedolPP, dehydrodolichyl diphosphate; E,E-FPP, E,E-farnesyl diphosphate; FARM, first aspartate-rich motif; GPP, geranyl diphosphate; IPP, isopentenyl diphosphate; UPP, undecaprenyl diphosphate; Z,E-FPP, Z,E-farnesyl diphosphate; Z,E-DecPP, Z,E-mixed decaprenyl diphosphate.
Trang 2showed that not only the primary but also the
three-dimensional structure of cis-prenyltransferase were
totally different from those of trans-prenyltransferases
[3-5] Homologous genes for cis-prenyltransferases
have been identified in various organisms [6–13],
revealing five highly conserved regions in the primary
structure of all cis-prenyltransferases [3] On the other
hand, cis-prenyltransferases are classified into three
subfamilies with respect to product chain length, i.e
short-chain (C15), medium-chain (C50)55), and
long-chain (C70)120) cis-prenyltransferases (Fig 1b)
Z,E-farnesyl diphosphate (Z,E-FPP, C15) synthase from
Mycobacterium tuberculosis, Rv1086, is the only
enzyme identified as a short-chain
cis-prenyltrans-ferase, which catalyses cis-condensation of one IPP
with geranyl diphosphate (GPP, C10) [11]
Medium-chain cis-prenyltransferases are represented by UPP
synthase which catalyses cis-condensation of eight IPPs
with E,E-FPP (C15) This enzyme is responsible for
biogenesis of undecaprenyl phosphate, an
indispens-able glycosyl carrier lipid in bacterial cell wall
biosyn-thesis Z,E-mixed decaprenyl diphosphate (Z,E-DecPP,
C50) synthase from M tuberculosis, Rv2361, is also
categorized in this subfamily [11] Most of dehydro-dolichyl diphosphate (DedolPP) synthases in eukaryo-tes catalysing synthesis of precursors of sugar carrier lipid dolichol during biosynthesis of glycoproteins, are categorized as long-chain cis-prenyltransferases Srt1p and Rer2p from Saccharomyces cerevisiae [7,8] and HDS from human [12] are included in this sub-family
One of the most interesting research topics on cata-lytic mechanisms of prenyl chain elongating enzymes
is to understand mechanisms by which individual pre-nyltransferases recognize prenyl chain lengths of allylic substrates and products Using a series of effective ran-dom chemical mutagenesis, Ohnuma et al showed that the ultimate product chain length of trans-prenyltrans-ferases was regulated by bulky residues located upstream of the first Asp-rich motif (FARM), which is one of the most highly conserved regions among trans-prenyltransferases [2,14,15] The bulky residues func-tion as a floor of the catalytic pocket for the growing isoprenoid chain to block further elongation Based on crystal structure and mutagenetic studies of the avian FPP synthase, Tarshis et al concluded that allylic
A
B
Fig 1 Classification of prenyltransferases (A) Schematic drawing of the reactions catalysed by trans-prenyltransferase and cis-prenyltrans-ferase (B) Classification of cis-prenyltransferases with respect to product chain lengths These enzymes catalyse cis-condensation of IPP (isoprene unit, C 5 ) onto an allylic diphosphate, all-E-prenyl diphosphate Grey lines indicate numbers of isoprene units of representative allylic substrates for each cis-prenyltransferase Red arrows indicate numbers of isoprene units of representative ultimate products, which are condensed with cis-configuration by each enzyme.
Trang 3diphosphate bound through Mg2+ to Asp residues in
the FARM motif [16] In contrast, mechanisms for
determination of the ultimate product chain length of
cis-prenyltransferases have not yet been determined
although mutational analysis of highly conserved
resi-dues and determination of crystal structures of UPP
synthase have enabled us to understand basic catalytic
mechanisms of cis-prenyltransferases [5,17–24]
In order to investigate regions important for
determination of product chain length in
cis-prenyl-transferases, we searched characteristic residues of
short- or long-chain cis-prenyltransferase subfamilies,
based on comparisons between primary structures of
cis-prenyltransferases identified, and crystal structures
of UPP synthases [5] Introduction of mutations in
regions of M luteus B-P 26 UPP synthase, which
correspond to characteristic residues, resulted in
dras-tic alteration of the ultimate product chain length of
prenyl products Amino acid residues located in close
proximity to the substrate binding site play an
essen-tial role in the synthesis of short-chain products such
as Z,E-FPP, while some charged residues on the
side-wall of the large hydrophobic cleft are important
to determine the ultimate chain length of polyprenyl
products These findings enabled us to understand
reaction mechanisms of cis-prenyltransferases, and
manipulate enzymes to produce various lengths of
Z,E-mixed polyisoprenoids
Results
In order to identify characteristic residues of short- or long-chain cis-prenyltransferase subfamilies, primary structures of proteins that were identified as cis-prenyl-transferases were compared Because Rv1086 from
M tuberculosis is the only enzyme cloned and identified as a short-chain cis-prenyltransferase, Z,E-FPP synthase [11], we attempted to identify Rv1086-specific residues that will help name the important amino acid residues for ultimate chain-length determin-ation As shown in Fig 2A, only Rv1086 possesses three Leu residues at positions 84, 85, and 90 instead
of the corresponding Ala, Phe, and Leu⁄ Trp in the conserved region III of other cis-prenyltransferases
Fig 2 Amino acid residues characteristic for short-chain
cis-prenyl-transferases functioning in the termination mechanism of cis-prenyl
chain elongation to produce short-chain Z,E-mixed prenyl
diphos-phates (A) Multiple alignment of amino acid sequences of
seven cis-prenyltransferases: Rv1086, Z,E-FPP synthase from
Mycobacterium tuberculosis (Accession No.; D70895); Rv2361c,
DecPP synthase from M tuberculosis (H70585); M luteus, UPP
synthase from M luteus B-P 26 (BAA31993); E coli, UPP synthase
from E coli (Q47675); Rer2p, DedolPP synthase from
Saccharo-myces cerevisiae (BAA36577); Srt1p, polyprenyl PP synthase from
S cerevisiae (NP_013819); HDS, DedolPP synthases from human
(BAC57588) Here, parts of sequences in conserved regions III and V
are shown Multiple alignment was obtained using the CLUSTAL W
program, and was edited using SeqVu Residues with more than
70% identity are boxed, and all residues are coloured as follows:
non-polar (G, A, V, L, I, P, F, M, W, C), yellow; uncharged non-polar (N, Q, S,
T, Y), green; acidic (D, E), red; basic (K, R, H), blue Red triangles
indicate residues that are characteristic for short-chain
cis-prenyl-transferase (B) TLC autoradiograms of polyprenyl alcohols derived
from products of cis-prenyl chain elongation with wild-type and
mutant UPP synthases, LL, LLL, F223H, and LLLH, using E,E-FPP
(left panel) of GPP (right panel) as allylic substrates Spots of
authentic standard prenyl alcohols are as follows: C 15 , E,E-farnesol;
C20, all-E-geranylgeraniol; C55, undecaprenol Ori; origin, S.F.; solvent
front.
Trang 4Furthermore, His-237 of Rv1086 downstream of the
conserved region V was found at the corresponding
position for Leu⁄ Phe residue in other
cis-prenyltrans-ferases
To investigate whether these Rv1086-characteristic
residues function in the reaction mechanism, we
replaced the corresponding residues of M luteus B-P
26 UPP synthase with these Rv1086-specific residues
to construct mutant enzymes with single-, double-,
triple-, and quadruple mutations, i.e F223H, A72L⁄
F73L (LL), A72L⁄ F73L ⁄ W78L (LLL), and A72L ⁄
F73L⁄ W78L ⁄ F223H (LLLH), respectively These
mutants were expressed in Escherichia coli, and
puri-fied to analyse prenyltransferase activity in vitro
Product distribution patterns of the mutants were
com-pared with those of the wild-type UPP synthase, which
produces C55 and C60 prenyl products in vitro The
TLC autoradiogram clearly showed that LL produced
shorter polyisoprenoids (C25)40 as major products),
and LLL produced even shorter polyisoprenoids
(C20)35 as major products) than wild-type UPP
syn-thase when E,E-FPP and GPP were used as allylic
sub-strates (Fig 2B)
In our previous study, site-directed mutagenesis at
the highly conserved Phe73 in region III of M luteus
B-P 26 UPP synthase resulted in a 32-fold increased
Km value for IPP, and a 16-fold decreased kcat value
[20] These results indicated that Phe73 in region III
was important for binding of IPP in the proper
direc-tion We also observed the shortening of the ultimate
chain length of prenyl products in mutants of
M luteus B-P 26 UPP synthase, i.e F73A and S74A
[20] To analyse functions of Ala72 and Trp78, which
are located in the vicinity of Phe73 in region III,
kin-etic constants of the mutants were determined A72L⁄
F73L showed an 88-fold higher Km value for IPP
compared with the wild-type enzyme However,
A72L⁄ F73L ⁄ W78L showed only a six-fold increased
Km value for IPP (Table 1), indicating that shortening
of the ultimate chain length of products by the triple
mutation, A72L⁄ F73L ⁄ W78L was not simply caused
by a decrease in the affinity of the catalytic domain for IPP In addition, effects of mutations at Ala72, Phe73, and Trp78 on Kmvalues for E,E-FPP were not signifi-cant (within threefold)
On the other hand, replacement of Phe223 with His in mutants F223H or LLLH, dramatically decreased catalytic activity when E,E-FPP was used
as allylic substrate (Fig 2B, left panel) However, when GPP was used, mutants showed certain cata-lytic activity producing C45-55 and C15 (Fig 2B, right panel) These results suggested that His237 of Rv1086, corresponding to Phe223 of M luteus B-P
26 UPP synthase, might function to prefer GPP as
an allylic substrate
We next investigated the characteristic residues of long-chain cis-prenyltransferase subfamilies to identify the important region for producing polyisoprenoid chains longer than C55 Multiple alignment of cis-pre-nyltransferases revealed that long-chain cis-prenyl-transferases such as Srt1p and Rer2p from
S cerevisiae [7,8] and HDS from human [12], have three to seven extra amino acid residues downstream
of the conserved region III (Fig 3A) This position corresponds to helix-3 of the M luteus B-P 26 UPP synthase, which participates in the constitution of the hydrophobic cleft The hydrophobic cleft is composed
of helix-2, helix-3, sheet-2, and sheet-4, and is consid-ered to accommodate the elongated prenyl intermedi-ates [5] In order to indicate the significance of these residues, we constructed two mutants of M luteus B-P 26 UPP synthase, i.e EKE and RAKDY, which contained insertions corresponding exactly to the extra amino acid residues of DedolPP synthases from human (HDS, positions 107–109) and yeast (Srt1p, positions 148–152), respectively (Fig 3A) Products from prenyltransferase reaction with these UPP syn-thase mutants in vitro were analysed by reversed-phase TLC As shown in Fig 3B, EKE produced relatively longer prenyl products with carbon chain lengths of C55)70 Furthermore, RAKDY gave even longer prenyl products with chain lengths of C60)75
In contrast, the control mutant with an insertion of five Ala residues instead of extra residues did not show such effects (Fig 3B) Introduction of extra amino acid residues caused a several times increase of
Km values for IPP (Table 1) In contrast, mutants EKE and RAKDY showed a 1.4–2.8-fold lower Km values for FPP, suggesting that the change in struc-ture of helix-3 moderately affected binding affinity for FPP
To confirm the significance of the extra resi-dues found in long-chain cis-prenyltransferases, we
Table 1 Kinetic constants for wild-type and mutant UPP synthases
of M luteus B-P 26.
Enzymes K m (IPP) (l M )a K m (FPP) (l M )
A72L ⁄ F73L ⁄ W78L (LLL) 49 ± 25 14 ± 2
a
For reactions with FPP.bPrevious data from [3].
Trang 5constructed a deletion mutant of Srt1p lacking the
five extra residues, Srt1p-delta (Fig 3A) Because
Srt1p expressed in E coli does not show significant
prenyltransferase activity, wild-type Srt1p and
Srt1p-delta were expressed in the yeast mutant strain
SNH23-7D which is deficient in the DedolPP synthase
gene, RER2, to analyse the reaction products clearly
Product analysis of the prenyltransferase reaction
with yeast crude proteins indicated that the mutant
Srt1p-delta produced shorter prenyl products with a
chain length of C65)95 as major products (Fig 3C),
while wild-type Srt1p produced C75)110 prenyl
prod-ucts as reported previously [8] Taken together, the
region in helix-3 is also very important for the
ulti-mate chain length determination mechanism for
cis-prenyltransferases
Discussion
In nature, a variety of Z,E-mixed polyisoprenoids with different carbon chain lengths from Z,E-FPP (C15) to natural rubber (C>10,000) are produced, and distribu-tion of carbon chain lengths of Z,E-mixed polyisopre-noids seems to depend on the origin of organisms Most bacteria produce only UPP (C55), while eukaryo-tes such as yeasts and mammals produce longer Z,E-mixed polyisoprenoids including DedolPP (C70)100)
In higher plants, various Z,E-mixed polyisoprenoids including dolichol and polyprenol are biosynthesized with two different distribution patterns of carbon chain length, encompassing C50)60 and C70)120 More-over, rubber producing plants such as Hevea brasilien-sisproduce high molecular weight cis-1,4-polyisoprene,
Fig 3 Extra amino acid residues
character-istic for long-chain cis-prenyltransferases are
important in producing long-chain Z,E-mixed
prenyl diphosphates (A) Multiple alignment
of amino acid sequences of seven
cis-pre-nyltransferases In this figure, a part of the
sequence of helix-3 is shown Similar
nota-tions as in Fig 2 are used for sequences
and colour usage The red upper line
indi-cates the extra residues that are
characteris-tic for long-chain cis-prenyltransferase.
Some parts of sequences of the mutants of
M luteus B-P 26 UPP synthase (EKE,
RAKDY) and of Srt1p (Srt1p-delta) are
shown (B) TLC autoradiograms of
polypre-nyl alcohols corresponding to products of
cis-prenyl chain elongation with wild-type
and mutant UPP synthases, EKE, RAKDY,
and AAAAA, using E,E-FPP as allylic
sub-strate C 55 indicates the spot of authentic
undecaprenol Ori, origin; S.F., solvent front.
(C) TLC autoradiograms of polyprenyl
alcohols corresponding to the products of
cis-prenyl chain elongation with wild-type
and mutants Srt1p, Srt1p-delta, using
E,E-FPP as substrate Spots of authentic
standard prenyl alcohols are as follows:
C15, E,E-farnesol; C55, undecaprenol; C75,
C75-polyprenol; C100, C100-polyprenol Ori,
origin; S.F., solvent front.
Trang 6i.e natural rubber Several lines of evidence suggest
that product chain lengths of cis-prenyltransferases can
be altered by environmental factors Yamada et al
[25,26] reported differences in chain lengths of dolichol
among tissues in the rat such as liver (C90)95) and
tes-tis (C85)90) Matsuoka et al [27] reported that chain
length distribution of products from DedolPP synthase
in microsomal fractions of rat liver could be affected
by reaction conditions in vitro such as detergents and
phospholipids However, recent reports on cloning and
characterization of cis-prenyltransferases from various
organisms showed that specificities of product chain
lengths mainly depended on different enzymatic
prop-erties attributable to structural diversity of each
enzyme Based on these facts, we investigated
charac-teristic residues of short- or long-chain
cis-prenyl-transferase subfamilies to elucidate chain length
determination mechanisms
Leu residues at positions 84, 85, and 90 in the
Z,E-FPP synthase Rv1086 were found to play a very
important role in shortening the ultimate chain length
of prenyl products (Fig 2) The corresponding residues
in M luteus B-P 26 UPP synthase, i.e Ala72, Phe73,
and Trp78, are located in the highly conserved region
III, and were suggested to be located close to the
bind-ing site for IPP, which had been identified by a series
of site-directed mutagenesis studies [20] In the crystal
structure of UPP synthase from M luteus B-P 26,
these residues are located at the edge of the large
hydrophobic cleft Recently, crystal structure of E coli
UPP synthase bound with the allylic substrate
E,E-FPP [28] has been revealed In this structure,
Ala69, which corresponds to Ala72 of M luteus B-P
26 UPP synthase, is located close to the x-end carbon,
C-14 of E,E-FPP at a distance of 3.2 A˚ More recently,
crystal structure of the D26A mutant E coli UPP
syn-thase, which shows about an 800-fold lower kcat value
than the wild-type enzyme, bound with IPP, has also
been described [29] In order to understand the
molecular mechanisms of chain length determination,
we built structural models of Rv1086 and of LLLH
mutant of M luteus UPP synthase using the E coli
UPP synthase-E,E-FPP complex structure as template,
based on multiple sequence alignments of
cis-prenyl-transferases previously identified Then, structures of
E,E-FPP and IPP were superimposed on the structural
models (Fig 4) In these proposed models, Leu residue
which corresponds to Ala69 of E coli UPP synthase is
closer to the C-14 of E,E-FPP, suggesting that the
bulky alkyl group of Leu72 in LLLH may interfere
with cis-addition of IPP onto E,E-FPP or Z,E-FPP
In contrast, Phe73 and Trp78 of M luteus B-P 26
UPP synthase, corresponding to Leu85 and Leu90 of
Rv1086, respectively, are not close to the binding site for E,E-FPP [5,28] In the crystal structure of
M luteusB-P 26 UPP synthase without substrates, res-idues from Ser74 to Val85 downstream of sheet-2 in the conserved region III could not be defined because
of high flexibility [5] In addition, substitution of the highly conserved Phe73 and Ser74 in region III of
M luteus B-P 26 UPP synthase into Ala resulted in 32- and 16-fold increases in Km value for IPP, and 16- and 12-fold decreases in kcat value, respectively [20] These results indicated that the flexible domain in the conserved region III was important for binding of IPP in the proper direction, and for catalytic function The double mutant A72L⁄ F73L prepared in the pre-sent study also showed an 88-fold higher Kmvalue for IPP compared with the wild-type enzyme However, the triple mutant A72L⁄ F73L ⁄ W78L showed a sixfold
Fig 4 Overall catalytic centre of a structural model for the LLLH mutant The model was built based on the crystal structure of E,E-FPP complex of UPP synthase from E coli (Protein Data Bank No 1V7U) Then, structures of E,E-FPP and IPP, which were identified from the complex structure of IPP and the D26A mutant of E coli UPP synthase (Protein Data Bank No 1X09), were superimposed
on the structural models Amino acid residues mutated in the LLLH mutant are indicated in red, and are overlapped with residues in the wild-type UPP synthase of M luteus B-P 26 The structural P-loop motif, proposed to function in binding of allylic substrates such as E,E-FPP, and charged residues including Arg197 and Arg203 are indicated.
Trang 7increased Km value for IPP (Table 1) These results
suggested that replacement of Trp78 for Leu was
necessary for constitution of a proper IPP binding
domain when Ala72 and Phe73 were replaced
with Leu residues which corresponded to Leu84, -85,
and -90, respectively, in Rv1086
Substitution of Phe223 with His dramatically
decreased the catalytic activity of UPP synthase when
E,E-FPP was used as allylic substrate However, GPP
could be accepted by these mutants as allylic substrate
to produce shorter prenyl products, i.e C45)55 and C15
(Fig 2B) In structural models of Rv1086 and the
LLLH mutant of M luteus UPP synthase (Fig 4),
His237 of Rv1086 is located in proximity of the
struc-tural P-loop motif, which is thought to recognize the
diphosphate group of allylic substrates such as
E,E-FPP [5,23] Substitution of His for Phe223 may
affect binding affinity for the allylic substrate with the
structural P-loop motif This hypothesis agrees with
the fact that in the prenyltransferase reaction by
Rv1086 in vitro, GPP and neryl diphosphate (C10) were
the only functional allylic substrates among the five
allylic substrates tested, including dimethylallyl
diphos-phate (C5) E,E-FPP and E,E,E-geranylgeranyl
diphos-phate (C20) [30]
On the other hand, three to seven extra amino acid
residues found downstream of the conserved region III
were shown to be important for the production of long
chain Z,E-mixed polyisoprenoids by the long-chain
cis-prenyltransferase subfamily According to the crystal
structure of UPP synthase of M luteus B-P 26, these
extra residues are located on the side wall of the large
hydrophobic cleft [5] Ko et al reported that
replace-ment of Leu137 of E coli UPP synthase, which is
located at the ‘bottom’ of the hydrophobic cleft, with
Ala resulted in elongation of the ultimate chain length
of Z,E-mixed polyisoprenoids, producing C55 and C60
as major products in the presence of 0.1% Triton
X-100 [21] Moreover, they indicated that the mutant
L137A produced C70 and C75 as major products in a
reaction without Triton X-100 for 96 h [21] Based on
these results, they proposed that Leu137 functioned as
the floor of the tunnel to block further elongation of
polyprenyl products This proposed model seems to be
analogous to the chain-length determination
mechan-ism of trans-prenyltransferases, in which bulky residues
located upstream of FARM play an important role in
determination of the ultimate prenyl chain length,
composing a suitable size for the pocket for growing
of the isoprenoid chain [2,15,16] According to this
model, we constructed and analysed a mutant of
M luteus B-P 26 UPP synthase L140A, which
corres-ponded to the E coli UPP synthase mutant L137A,
and found that the mutant also produced C55and C60
as major products in the presence of Triton X-100 (data not shown) However, our results obtained in the present study couldn’t be explained by the model pro-posed by Ko et al because Leu137 of E coli UPP synthase did not correspond to the domain where we introduced the extra amino acid residues, and the mutants EKE and RAKDY produced even longer polyprenyl products (C60)75) than the E coli UPP syn-thase mutant L137A in the presence of Triton X-100 Insertion of five Ala residues instead of the peptides RAKDY did not cause a significant change in the length of prenyl products, indicating a requirement for insertion of specific amino acid residues for lengthen-ing the ultimate product chain Moreover, this sugges-ted the significance of some charged or polar residues
at the proper positions rather than expansion of the interior space of the hydrophobic cleft
In the crystal structure of E coli UPP synthase bound with E,E-FPP, helix-3 is kinked to be closer to the E,E-FPP binding domain compared with the struc-ture without substrates [28], indicating that the strucstruc-ture
of UPP synthase shifts to the closed conformation when the substrate binding site is shared with the allylic sub-strate The interior of the large hydrophobic cleft, sur-rounded by sheet-2, sheet-4, helix-2, and helix-3, mainly consists of hydrophobic residues [5] Hydrophobic resi-dues on kinked helix-3 in the closed conformation may function as a guide rail to introduce the elongating pre-nyl chain in the proper direction (Figs 5A and B) Although most of residues constituting the hydrophobic cleft are highly conserved among cis-prenyltransferases, residues localized on helix-3 show a wide diversity (Fig 3A) Moreover, the domain in which we intro-duced extra amino acid residues corresponded to the hinge region of the kinked helix-3 (Fig 5B) Therefore,
we proposed that charged residues inserted at the hinge region of helix-3 might control the bending direction of the growing hydrophobic prenyl chain along the hydro-phobic interior of helix-3 so that the hydrohydro-phobic cleft could accommodate the bulk of the prenyl chain to fit a suitable size during enzymatic elongation
In conclusion, we identified critical regulatory domains in cis-prenyltransferases for determination of the ultimate product chain length, and proposed a model for chain-length determination Further elucida-tion and manipulaelucida-tion of chain-length determinaelucida-tion mechanisms of cis-prenyltransferase are not only attractive but also very important as a biotechnological aspect because biological materials composed of the polymer of IPP with cis-configuration such as natural rubber, can be used for the development of novel func-tional materials
Trang 8Experimental procedures
Materials and general procedures
Nonlabelled IPP and E,E-FPP were synthesized according
to Davisson et al [31] 1-14C-labelled IPP (1.95 TBqÆmol)1)
was from Amersham Biosciences (Princeton, NJ, USA)
Restriction enzymes and other DNA modifying enzymes
were from TaKaRa Bio (Otu, Japan) and TOYOBO
(Osaka, Japan) Potato acid phosphatase was from Sigma
Precoated reversed phase TLC plate, LKC-18 was
pur-chased from Whatman (Brentford, UK) The yeast strain
SNH23-7D, mutant allele rer2-2 [7], and pRS316 containing
SRT1[8] were kindly provided by A Nakano and M Sato
(RIKEN, Japan) The expression vector for yeast, pJR1133
was kindly provided by A Ferrer (University of Barcelona,
Spain) Restriction enzyme digestions, transformations, and
other standard molecular biological techniques were carried
out as described by Sambrook et al [32] All other
chemi-cals were of analytical grade
Multiple alignments and homology modelling
of mutant enzymes
Amino acid sequences of cis-prenyltransferases already
identified were aligned using the clustal w Multiple
Sequence Alignment Program [33] The resulting alignment
was edited by seqvu (Shareware alignment programme,
Garvan Institute, Sydney, Australia) Three-dimensional models for the mutants, Rv1086 and Srt1p were obtained
by The swiss-model alignment interface (http://swissmodel expasy.org/) using multiple alignment of cis-prenyltrans-ferases and crystal structures of UPP synthase from
M luteus B-P 26 [5] or E coli [21,28,29] as structural templates
Expression vector system and site-directed mutagenesis
The expression plasmid, pMluUEX for M luteus B-P 26 UPP synthase [20] was used as template for preparation of the mutants Site-directed mutagenesis was carried out according to protocols for the Gene Editor in vitro Site-Directed Mutagenesis System (Promega, Madison, MI, USA) The single stranded wild-type UPP synthase gene used
as template in the mutagenesis reaction was prepared by infection of E coli JM109 cells (TaKaRa) harboring pMluUEX with R408 helper phages Mutagenic oligonucleo-tides designed to produce the desired mutant enzymes were: 5¢-CAGTTGACAATAAGTACAGCG-3¢ (for A72L ⁄ F73L); 5¢-CTTTAGGTCGACTCAAATTTTCAGTTGACAATAA GTACAGCG-3¢ (for A72L ⁄ F73L ⁄ W78L); 5¢-CCGGCCAG TGTTCATCGATAAATAC-3¢ (for F223H); 5¢-CTTTAGG TCGACTCAAATTTTCAGTTGACAATAAGTACAGCG-3¢ and 5¢-CCGGCCAGTGTTCATCGATAAATAC-3¢ (for A72L⁄ F73L ⁄ W78L ⁄ F223H) For insertion of three or five
Fig 5 Large hydrophobic cleft of M luteus B-P 26 UPP synthase with or without FPP Models were built based on the crystal structure of E,E-FPP complex of UPP synthase from E coli (Protein Data Bank No 1V7U) Then, the structure of E,E-FPP was superimposed on the models Side chains are coloured as follows: nonpolar, grey; uncharged polar, yellow; acidic, red; basic, blue (A) Large hydrophobic cleft of
M luteus B-P 26 UPP synthase (Protein Data Bank No 1F75) consisting of helix-2, helix-3, sheet-2, and sheet-4 Only side chains facing towards the inside of the hydrophobic cleft are shown (B) Structural model of the large hydrophobic cleft of M luteus B-P 26 UPP synthase with E,E-FPP Only side chains facing towards the inside of the hydrophobic cleft are shown The white arrow indicates a predicted direction
of the prenyl chain elongation The circle indicates regions in which the extra amino acid residues, EKE or RAKDY were introduced.
Trang 9amino acid residues between Pro101 and Glu102 of M luteus
B-P 26 UPP synthase, oligonucleotides used were: 5¢-CAAT
GAGTTCCTCCTTCTCCGGTAAAAATGTG-3¢ (for EKE);
and 5¢-AACATTTTTTTCAATGAGCTCATAGTCCTTG
GCTCTCGGTAAAAATG-3¢ (for RAKDY) Introduction
of mutations was confirmed by sequencing whole nucleotide
sequences using the dideoxy chain-termination method with
a DNA sequencer (LI-COR, model 4200, ALOKA, Tokyo,
Japan)
Construction of deletion mutant for Srt1p
The SRT1 expression plasmid was constructed with the
SRT1fragment amplified by PCR using appropriate
prim-ers The sense and antisense primers, 5¢-CGTTTCTGGGT
ACCATAATGAAAATGC-3¢ and 5¢-CTAGTTGTCGACT
TTTACTTATTCATC-3¢, respectively, were designed to
create a KpnI site upstream the starting codon ATG, and a
SalI site downstream the stop codon TAA, respectively
The PCR product was digested with KpnI and SalI, and
separated on a 1% agarose gel The desired band was
elut-ed, and ligated into the pBluescript II SK(–) vector, then
digested with the same restriction enzymes to give
pBS-Srt1 Five amino acids deletion construct of Srt1p
(Srt1p-delta) was generated using the Gene Editor in vitro
Site-Directed Mutagenesis System (Promega) with the
primer 5¢-GATGAATTCGCGAAGAAGGATCCCTTAT
AC-3¢ to obtain pBS-Srt1p-delta Wild-type SRT1 and
the mutant Srt1p-delta were digested with KpnI and SalI,
subcloned into pJR1133 vector, and digested with the same
restriction enzymes to give Srt1p and
pJR1133-Srt1p-delta, respectively
Overproduction and purification of UPP synthase
mutant
Each construct for expression of target proteins was
introduced into E coli BL21(DE3) cells, and cells were
cultured in Luria–Bertani or M9YG medium The
proce-dures us for overproduction and purification of UPP
syn-thase mutants and the wild-type enzyme were essentially
similar to those described in a previous paper [20,24]
Purity of the mutant enzymes was analysed by
SDS⁄ PAGE with Coomassie brilliant blue staining
Pro-tein concentrations were measured by the method of
Bradford with BSA as standard
Expressions of Srt1p and Srt1p-delta in yeasts
pJR1133-Srt1p and pJR1133-Srt1p-delta plasmids were
introduced into yeast rer2-2 mutant strain, SNH23-7D
(MATa rer2 mfa1::TRP1::HIS3 ura3 trp1 ade2 leu2 his3
lys2), which is deficient in the activity of DedolPP synthase
[8] Transformants were selected as Ura+ colonies, were
cultured at 23C on agar plates containing minimal medium (0.16% yeast nitrogen base without amino acids, 0.5% ammonium sulfate, 2% glucose, supplemented with
60 lgÆmL)1 Leu and 30 lgÆmL)1 Lys) Yeast cells were grown in 100 mL minimal medium until the late-logarithmic phase (OD600of 1.0)
Preparation of yeast membrane fraction proteins from wild-type Srt1p and Srt1p-delta mutants Yeast cells were harvested by centrifugation at 3000 g for
5 min, suspended in 300 lL zymolyase buffer (50 mm Tris⁄ HCl pH 7.5, 10 mm MgCl2, 1 m sorbitol, 30 mm dithiothreitol, 2 mgÆmL)1 zymolyase 100T), and incubated
at 30C for 30 min The spheroplast-formed cells were collected by centrifugation at 1000 g for 5 min, and resus-pended in 400 lL breakage buffer (50 mm KH2PO4
pH 7.5, 1 m dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 1 lgÆmL)1 aprotinin, 1 lgÆmL)1 leupeptin, and
1 lgÆmL)1 pepstatin A) Using an equal volume of glass beads, cell suspensions were vortexed for 30 s, followed by incubation in an ice bath for 1 min, repeated five times Cell lysates were centrifuged at 300 g for 5 min to remove unbroken cells Supernatants were further centrifuged at
13 000 g for 10 min to separate membrane and soluble fractions
Cis-Prenyltransferase assay and product analysis UPP synthase activity was measured by determining amounts of [1-14C]IPP incorporated into butanol-extracta-ble polyprenyl diphosphates The standard assay mixture contained 100 mm Tris⁄ HCl pH 7.5, 0.5 mm MgCl2,
10 lm E,E-FPP, 10 lm 1-14C-labelledIPP (37 MBqÆmol)1), 0.05% (w⁄ v) Triton X-100, and a suitable amount of enzyme solution in a final volume of 200 lL For the yeast cis-prenyltransferase assay, the standard reaction mixture contained (final volume of 100 lL) 25 mm phos-phate buffer pH 7.5, 20 mm 2-mercaptoethanol, 20 mm potassium fluoride, 4 mm MgCl2, 50 lm [1-14C]IPP,
10 lm E,E-FPP, and 50 lg proteins of membrane frac-tions After incubation at 30C for 30 min, reaction products were extracted with 1-butanol saturated with water, and radioactivity in the butanol extract was meas-ured with an Aloka LSC-1000 liquid scintillation counter For product analysis, radioactive prenyl diphosphate products in the reaction mixture were hydrolysed to the corresponding alcohols with potato acid phosphatase according to the method of Fujii et al [34] Product alcohols were extracted with pentane, and analysed by reversed phase TLC with a solvent system of acet-one⁄ water (19 : 1) (for bacterial cis-prenyltransferase) or acetone⁄ water (39 : 1) (for yeast cis-prenyltransferase) Positions of authentic standards were visualized with
Trang 10iodine vapour, and distribution of radioactivity was
ana-lysed with a Fuji BAS 1000 Mac Bioimage Analyzer
Acknowledgements
We are grateful to Dr A Nakano and Dr M Sato
(RIKEN, Japan) for kindly providing the yeast strain
SNH23-7D, and Dr A Ferrer (University of
Barce-lona, Spain) for kindly providing the vector pJR1133
This work was supported in part by Grants-in-Aid
for Scientific Research from JSPS, Japan Society for
the Promotion of Science, and by the Foundations of
Takeda Science, Eno Science, The Yuasa International,
and by the Sumitomo Foundation
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