跳到主要內容

臺灣博碩士論文加值系統

(44.212.99.208) 您好!臺灣時間:2024/04/17 20:52
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:陳姵君
研究生(外文):Chen Annie Pei-Chun
論文名稱:大腸桿菌十一異戊二烯焦磷酸合成酶之反應機制及動力學研究:其在發展抗菌藥物之應用
論文名稱(外文):Mechanistic and Kinetic Studies of E. coli Undecaprenyl Pyrophosphate Synthase : Application in Antibacterial Drug Discovery
指導教授:梁博煌
指導教授(外文):Po-Huang Liang
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
畢業學年度:93
語文別:英文
論文頁數:140
中文關鍵詞:十一異戊二烯焦磷酸合成酶動力學研究類異戊二烯族
外文關鍵詞:Undecaprenyl Pyrophosphate synthaseisoprenyl pyrophosphate synthasekinetic studyantibacterial drug target
相關次數:
  • 被引用被引用:0
  • 點閱點閱:211
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
類異戊二烯族化合物廣泛分布於自然界,是由異戊二烯焦磷酸構成的聚合物。此類化合物的生合成是經由一類異戊二烯轉移酵素所催化。這類酵素催化多個含5個碳異戊二烯焦磷酸和含15個碳法呢基焦磷酸結合生成長鏈產物。這些酵素扮演著重要的生理功能,例如: 十一異戊二烯焦磷酸合成酶產生的55個碳的產物,可攜帶醣質以合成細菌胞壁,因此,可藉由研發此酵素之抑制劑來做為抗生素藥物。我們先前已探討此酵素的反應機制與立體結構,並且利用定點突變的方法,對於此酵素參與活化反應的氨基酸與受質結合的位置,及其催化反應中的速率決定步驟,有初步了解。本篇論文首先探討此酵素調控其產物(為合成細菌胞璧的前趨物)鏈長生成之重要氨基酸,並應用合成此酵素受質的螢光類似物,觀察酵素反應進行中螢光的變化,更進一步利用阻流反應分析儀,探討此受質螢光類似物與酵素的細部反應機制。 同時,根據受質的鏈長及磷酸根與活化區域的構型,找出此酵素對於與受質及其生成之長鏈產物的專一性。此外,我也探討二價鎂離子對於十一異戊二烯焦磷酸聚合反應的重要性,而異戊二烯焦磷酸與酵素的結合是藉由與鎂離子形成複合體,再進入活化位置與法呢基焦磷酸進行催化反應。最後,更希望利用法呢基焦磷酸螢光類似物能聚合生成長鏈產物的特性,應用於抗生素藥物篩選。 希望藉由本篇論文對十一異戊二烯焦磷酸詳盡及完整的研究,成為對此類異戊二烯轉移酵素的模版與研發抗生素藥物的指標。
Farnesyl pyrophosphate (FPP) serves as a branch point to synthesize a variety of
natural isoprenoids. Undecaprenyl pyrophosphate synthase (UPPs) is one member of
the prenyltransferases, catalyzes the consecutive cis- condensation reactions of a
farnesyl pyrophosphate (FPP) with eight isopentenyl pyrophosphates (IPP), to
generate undecaprenyl pyrophosphate (UPP) that serves as a lipid carrier for
peptidoglycan synthesis of bacterial cell wall. Because of its pivotal role in cell wall
biosynthesis, the enzyme is essential for bacterial survival and could be regarded as an
antibiotic drug target. Previously, we have examined the pre-steady state analysis of
multiple-step UPPs reaction in terms of IPP condensation, and determined the product
release as the rate of determining step during catalysis. Here, the detail mechanistic
and kinetic studies of E. coli undecaprenyl pyrophosphate synthase, as well as its
application in antibacterial drug discovery would be investigated in this thesis.
UPPs’ substrate binding and protein conformational change during reaction were
determined via site directed mutagenesis, fluorescence quenching and stopped-flow
methods. With the aid of the fluorescent analogue of FPP (TFMC-GPP), UPPs binds
FPP in a rapid equilibrium manner was determined to have a fast release rate constant
of 30 s-1, and the product dissociation rate constant of 0.5 s-1 from the competition
experiments. Furthermore, during UPPs catalysis, a three-phase protein fluorescence
change with time was observed in stopped-flow apparatus. Another synthesized FPP
analogue, Farnesyl thiolopyrophosphate (FsPP), with a much less labile
thiolopyrophosphate, serves as a poor substrate, and suggested that the first phase is
due to the IPP binding to E•FPP complex and the other two slow phases are originated
from the protein conformational change which coincided with the time course of FPP
chain elongation from C15 to C55 and product release, respectively. Also, our
fluorescence quenching data indicate the binding order of substrates to UPPs, such
that FPP needed to be first bound to the active site prior to IPP. In addition,
fluorescent analogues of FPP, compounds containing amide- or ester-linked
N-methylanthraniloyl group display fluorescence resonance energy transfer with
UPPs and receive emitted fluorescence from Trp31, Trp75, Trp91, and Trp221
residues in close proximity. These two probes were utilized to study the active site
conformation and topology of E. coli UPPs. Finally, the UPPs intrinsic fluorescence
quenched upon FPP binding mainly due to quenching the fluorescence of Trp91, a
residue in the α3 helix that moves toward the active site during substrate binding.
This data later was found agreeable to the crystal structure.
From our reported the co-crystal structure of E. coli UPPs in complex with FPP,
its phosphate head-group is bound to the positively-charged Arg residues and the
hydrocarbon moiety interacts with hydrophobic amino acids including Leu85, Leu88,
and Phe89, located on the α3 helix of UPPs. We further determine the role of
pyrophosphate, and demonstrated the importance of pyrophosphate in terms of
substrate allocation. A mono-phosphate analogue of FPP binds UPPs with a 8-fold
lower affinity (Kd=4.4 μM) compared with the pyrophosphate analogue, a result of a
larger dissociation rate constant (koff=192 s-1), whereas farnesol (1 mM) lacking the
pyrophosphate does not inhibit the UPPs reaction. Moreover, Geranylgeranyl
pyrophosphate (GGPP) containing a larger C20 hydrocarbon tail or a shorter C10 GPP
both serve equally good substrates (similar kcat value) compared with FPP.
Replacement of Leu85, Leu88, or Phe89 with Ala increases FPP and GGPP Km values
by the same amount, indicating that these amino acids are important for substrate
binding, but do not determine substrate specificity. Regardless the substrate with a
shorter of longer hydrocarbon tail, UPPs would still catalyze eight IPP condensation
reactions to generate product that could accommodate in the large upper portion of
active site. Besides computer modeling data suggested the importance of the residues
positioned at the upper portion of active site in product chain length determination,
data suggested that the small side chain of Ala69 is required for rapid elongation to
the C55 product, while the large hydrophobic side chain of Leu137 is required to limit
the elongation to the C55 product. Furthermore, the roles of residues Ser71, Glu73,
Asn74, Trp75, Arg77 and Glu81 located on a flexible loop attached to α3 helix were
investigated. The loop may function to bridge the interaction of IPP with FPP, needed
to initiate the condensation reaction and serve as a hinge to control the substrate
binding and product release.
Unlike trans-type prenyltransferase, in cis-type UPPs, no DDXXD motif was
found. As the fluorescence binding study showed that FPP binding did not require
Mg2+, whereas IPP binding and the ensuring reactions absolutely required the metal
ion, the role of metal was further studied. In the co-crystal structure of wild-type
enzyme with divalent metal ion, Mg2+ is coordinated by the pyrophosphate of FsPP,
the carboxylate of Asp26, and three water molecules. In case of Asp26 mutated to
alanine, Mg2+is bound to the pyrophosphate of IPP. The role of Asp26 is likely to
assist the migration of Mg2+ from IPP to FPP, and thus initiating the condensation
reaction by ionization of the pyrophosphate group from FPP. The [Mg2+] dependence
of the catalytic rate by UPPs shows that the activity is maximal at [Mg2+] = 1 mM, but
drops significantly when Mg2+ ions are in excess (50 mM). Without Mg2+, IPP binds
to UPPs only at high concentration. Notably, substitutions of other divalent metal ions
were not able to compensate the role of Mg2+. Other conserved residues including
His43, Ser71, Asn74 and Arg77 may serve as general acid/base and pyrophosphate
carrier.
In summary, our results provide a thorough understanding for E. coli UPPs in its
mechanistic and kinetic studies, in terms of protein conformational change, substrate,
and product specificity, product chain length determination, role of flexible loop, and
role of metal during catalysis. We have proposed a catalytic mechanism of UPPs, as
well as an overall protein conformational change in reaction. Not only this study
could be a model protein for understanding family of prenyltransferases, but it also
provides a good target system for further identification of anti-bacterial drug
discovery.
TABLE OF CONTENTS
摘 要 vi
ABSTRACT vii
LIST OF SCHEME & TABLE xi
LIST OF FIGURE xii
ABBREVIATION xiv
1. INTRODUCTION
1.1 Isoprenoids 1
1.2 Classification of prenyltransferases 1
1.3 Isoprenyl pyrophosphate synthase 2
1.4 Trans-type prenyltransferases 4
1.5 Cis-type prenyltransferases: undecaprenyl pyrophosphate synthase 4
1.5.1 Reaction mechanism: product distribution and rate of condensation 6
1.5.2 Substrate binding site 9
1.5.3 Cis-type UPPs crystal structures 9
1.6 Specific aim 10
2. MATERIAL AND METHODS
2.1 Chemicals 14
2.2 Site-directed mutagenesis of UPPs 15
2.3 Purification of His-tagged UPPs and removal of tag 17
2.4 Kinetic constant measurements 19
2.4.1 Kinetic constant measurements of Km and kcat values 19
2.4.2 Kinetic measurements of synthesized compound as alternative substrate 20

2.4.3 Measurement of inhibition constant of synthesized compound 21
2.4.4 Enzyme activity assay in various concentration of Triton X-100 21
2.4.5 Reaction kinetics of UPPs with various concentrations of Mg2+ and different metal ions 22
2.5 The final products formation and analysis 23
2.5.1 Identification of reaction products of TFMC-GPP with IPP 24
2.6 Single-turnover reaction of UPPs using GGPP, or MANT-ester-GPP, MANT-amide-GPP as substrate 24
2.7 Photospectroscopies and quantum yield of synthesized compound 25
2.8 Fluorescence spectrophotometer assay: monitor the
conformational change of UPPs 25
2.8.1 Role of Mg2+ in substrate binding to UPPs 26
2.9 Fluorescence monitoring of fluorescent probe 27
2.9.1 Fluorescence titration of TFMC-GPP with UPPs and FPP and stoichiometry determination 27
2.9.2 Fluorescence titration of TFMC-GP with UPPs and FPP 27
2.9.3 Fluorescence titration of MANT-ester-GPP and MANT-amide-GPP with UPPs and FPP 28
2.9.4 Fluorescence resonance energy transfer titration spectra of MANT-ester-GPP 28
2.10 Stopped-flow experiments 28
2.10.1 Monitor the protein conformational change during catalysis 29

2.10.2 Measurements of kon and koff of substrate analogue 29
2.11 Circular dichroism (CD) Experiments 31
3. RESULTS
3.1 Mechanism of product chain length 32
3.1.1 Products generated by I62A, H103A, V105A, and L137A UPPs 32
3.1.2 The roles of amino acid residues in the disordered loop of 72–83 34
3.2 Probing the conformational change during catalysis using an inhibitor and tryptophan mutants 36

3.2.1 FsPP is an inhibitor and an alternative substrate for UPPs 36
3.2.2 Site-directed mutagenesis and kinetic parameters of the mutant UPPs 38
3.2.3 FPP quenches the UPPs intrinsic fluorescence 38
3.2.4 IPP increases the fluorescence of binary UPPs•FPP (FsPP) complex 42
3.2.5 Monitoring of protein conformational change using stopped-flow experiments 42

3.3 Substrate and product dissociation kinetics 46
3.3.1 Spectroscopic characterization of TFMC-GPP 47
3.3.2 TFMC-GPP serves as inhibitor and alternative substrate for UPPs 50
3.3.3 Fluorescence titration of TFMC-GPP with UPPs and FPP 52
3.3.4 Kinetics of TFMC-GPP association and dissociation 52
3.3.5 Substrate and product dissociation kinetics 55
3.4 High concentration of Triton X-100 inhibits the enzyme activity 57
3.5 Substrate specificities 57
3.5.1 Role of pyrophosphate of the allylic substrate in binding with UPPs 57
3.5.2 Role of hydrocarbon moiety of allylic substrate in UPPs reaction 61
3.5.3 L85, L88 and F89 are essential in binding substrate but not important in distinguishing GGPP from FPP 64
3.5.4 Products of C20-GGPP and IPP under steady-state and single-turnover conditions 66
3.6 Role of metal ions 68
3.6.1 Binding mode studied by fluorescence experiments 68
3.6.2 Reaction kinetics with different concentrations of Mg2+ 70
3.6.3 Protein stability and secondary structure measured by CD 73
3.7 Probing fluorescence energy transfer 73
3.7.1 Spectroscopic features of N-methylisotic-labeled FPP analogue 76
3.7.2 Fluorescence titration of MANT-ester-GPP and MANT-amide-GPP with UPPs and FPP 78
3.7.3 Binding of MANT-ester-GPP with UPPs determined using stopped-flow experiments 79
3.7.4 Study of fluorescence resonance energy transfer for UPPs 82
3.7.5 Identification of Trp residues contributing FRET to MANT-ester-GPP 82
3.7.6 MANT-ester-GPP and MANT-amide-GPP serve as alternative substrates for UPPs 85
3.8 Application of MANT-ester-GPP in drug screening 91
4. DISCUSSION
4.1 Mechanism of product chain length 94
4.2 Identification of active conformation and role of flexible loop 95
4.3 Probing the conformational change during catalysis using an inhibitor and tryptophan mutants 101

4.4 Study of ligand interactions via a fluorescent substrate analogue 103
4.5 Role of Triton X-100 during catalysis 106
4.6 Substrate binding mode 108
4.7 Substrate and product specificities 110
4.8 Rationale of product specificities 112
4.9 Role of the metal ion in UPPs catalysis 114
4.10 UPPs reaction and proposed catalytic mechanism 118
4.11 Characterization of environmental sensitive N-methylisotic-labeled FPP analogue 123

4.12 Probe fluorescence energy transfer and inhibitor binding for UPPs 124
4.13 Application in drug discovery 127
5. CONCLUDING REMARK 128
REFERENCE 130
APPENDIX (LIST OF PUPLICATION) 141
[1] Liang, P. H., Ko, T. P., & Wang, A. H.-J. (2002) Structure, mechanism, and
function of prenyltransferases. Eur. J. Biochem. 269, 3339-3354.
[2] Poulter C. D. & Rilling, H. C. (1981) Prenyl transferase and isomerase. In
Biosynthesis of isoprenoid compounds (Spurgeon, SL, R., ed.) Vol. 1, pp 161-224,
John Wiley & Sons, New York.
[3] Poulter, C. D. (1974) Model studies in terpene biosynthesis. J. Agric. Food Chem.
22,167-173.
[4] Poulter C. D. & Rilling, H. C. (1981) Conversion of farnesyl pyrophosphate to
squalene. In Biosynthesis of isoprenoid compounds (Spurgeon, SL, R., ed.) Vol. 1, pp
225-282, John Wiley & Sons, New York.
[5] Spurgeon, S. L. & Poulter, C. D. (1981) Biosynthesis of carotenoid. In
Biosynthesis of isoprenoid compounds (Spurgeon, SL, R., ed.) Vol. 2, pp 1-112, John
Wiley & Sons, New York.
[6] Sinensky, M. (2000) Recent advances in the study of prenylated proteins. Biochim
Biophys Acta 1484, 93-106.
[7] Gelb, M. H., Scholten, J. D. & Sebolt-Leopold J. S. (1998) Protein prenylation:
from discovery to prospects for cancer treatment. Curr. Opin. Chem. Biol. 2, 40-48.
[8] Kellogg, B. A. & Poulter C. D. (1997) Chain elongation in the isoprenoid
biosynthetic pathway. Curr. Opin. Chem. Biol. 1, 570-578.
[9] Clarke, C. F. (1992) Protein isoprenylation and methylation of at
carboxyl-terminal cysteine residue. Annu. Rev. Biochem. 61, 355-386.
[10] Glomset, J. A. & Farnsworth, C. C. (1994) Role of protein modification reactions
in programming interactions between Ras-related FTPases and cell membranes. Annu.
Rev. ell Biol. 10, 181-205.
[11] Schafer, W. R. & Rine, J. (1992) Protein prenylation: genes, enzymes, targets and
function. Annu. Rev. Genet. 26, 209-237.
[12] Wendt, K. U., Poralla, K. & Schulz, G. E. (1997) Structure and function of a
squalene cyclase. Science 275, 1811-1815.
[13] Rynkiewicz, M. J., Cane, D. E., & Christianson, D. W. (2001) Structure of
trichodiene synthase from Fusarium sporotrichioides provides mechanistic inferences
on the terpene cyclization cascade. Proc. Natl. Acad. Sci. U.S.A. 98, 13543-13548.
[14] Ogura, K., Koyama, T., & Sagami, H. (1997) Polyprenyl diphosphate synthases,
Subcellular Biochem. 28, Chapter 3, 57–88.
[15] Ogura, K. & Koyama, T. (1998) Enzymatic aspects of isoprenoid chain
elongation. Chemical Reviews 98, 1263-1276.
[16] Chen, A., Kroon, P. A., & Poulter, C. D. (1994) Isoprenyl diphosphate synthases:
protein sequence comparisons, a phylogenetic tree, and predictions of secondary
structure. Protein Sci. 3, 600-607.
[17] Wang, K.C., & Ohnuma, S.-I. (2000) Isoprenyl diphosphate synthases. Biochim.
Biophys. Acta 1529, 33-48.
[18] Ohnuma, S.-I., Suzuki, M. & Nishino, T. (1994) Archaebacterial ether-linked
lipid biosynthetic gene. Expression cloning, sequencing and characterization of
geranylgeranyl-diphosphate synthase. J. Biol. Chem. 269, 14792-14797.
[19] Chen, A. & Poulter, C. D. (1994) Isolation and characterization of idsA: the gene
for the short chain isoprenyl diphosphate synthase from Methanolbacterium
thermoautotrophicum. Arch. Biochem. Biophys. 314, 399-404.
[20] Tachibana, A., Yano, Y. Y., Otani, S., Nomura, N., Sako, Y, & Taniguchi, M.
(2000) Novel prenyltransferase gene encoding farnesylgeranyl diphosphate synthase
from a hyperthermophilic archeon, Aeropyrum pernix. Eur J. Biochem. 267, 321-328.
[21] Asai, K.-I., Fujisaki, S., Nishimura, Y., Nishino, T., Okada, K., Nakagawa, T.,
Kawamukai, M. & Matsuda, H. (1994) The identification of Escherichia coli ispB (cel)
gene encoding the octaprenyl diphosphate synthase. Biochem. Biophys. Res.
Commun. 202, 340-345.
[22] Okada, K., Suzuki, K., Kamiya, Y., Zhu, X., Fujisaki, S., Nishimura, Y., Nishino,
T., Nakagawa, T., Kawamukai, M. & Matsuda, H. (1996) Polyprenyl diphosphate
synthase essentially defines the length of the side chain of ubiquinone. Biochim.
Biophys. Acta 1302, 217-223.
[23] Okada, K., Minehira, M., Zhu, X., Suzuki, K., Nakagawa, T., Matsuda, H. &
Kawamukai, M. (1997) The ispB gene encoding octaprenyl diphosphate synthase is
essential for growth of Escherichia coli. J. Bacteriol. 179, 3058-3060.
[24] Ohnuma, S.-I., Koyama, T. & Ogura, K. (1992) Chain length distribution of the
products formed in solanesyl diphosphate synthase reaction. J. Biochem. (Tokyo) 112,
743-749.
[25] Ohnuma, S.-I., Koyama, T. & Ogura, K. (1991) Purification of
solanesyl-diphosphate synthase from Micrococcus luteus. A new class of
prenyltransferase. J. Biol. Chem. 266, 23706-23713.
[26] Teclebrhan, H., Olsson, J., Swiezewska, E. & Dallner, G. (1993) Biosynthesis of
the side chain of ubiquinone: trans-prenyltransferase in rat liver microsomes. J. Biol.
Chem. 268, 23081-23086.
[27] Suzuki, K., Okada, K. Kamiya, Y., Zhu, X. F., Nakagawa, T., Kawamukai, M. &
Matsuda, H. (1997) Analysis of the decaprenyl diphosphate synthase (dps) gene in
fission yeast suggests a role of ubiquinone as an antioxidant. J. Biochem. (Tokyo)
121, 496-505.
[28] Koike-Takeshita, A., Koyama, T., Obata, S. & Ogura, K. (1995) Molecular
cloning and nucleotide sequences of the genes for two essential proteins constituting a
novel enzyme system for heptaprenyl diphosphate synthesis. J. Biol. Chem. 270,
18396-18400.
[29] Ashby, M. N. & Edwards, P. A. (1990) Elucidation of the deficiency in two yeast
coenzyme Q mutants. Characterization of the structural gene encoding hexaprenyl
pyrophosphate synthetase. J. Biol. Chem. 265, 13157-13164.
[30] Shimizu, N., Koyama, T. & Ogura, K. (1998) Molecular cloning, expression, and
characterization of the genes encoding the two essential protein components of
Micrococcus luteus B-P 26 hexaprenyl diphosphate synthase. J. Bacteriol. 180,
1578-1581.
[31] Collin, M. D. & Jones, D. (1981) Distribution of isoprenoid quinone structural
types in bacteria and their taxonomic implication. Microbiol. Rev. 45, 316-354.
[32] Oh, S. K., Han, K. H., Ryu, S. B. & Kang, H. (2000) Molecular cloning,
expression, and functional analysis of a cis-prenyltransferase from Arabidopsis
thaliana. J. Biol. Chem. 275, 18482-18488.
[33] Shimizu, N., Koyama, T., & Ogura, K. (1998) Molecular cloning, expression,
and purification of undecaprenyl diphosphate synthase. J. Biol. Chem. 273,
19476–19481.
[34] Apfel, C. M., Takacs, B., Fountoulakis, M., Stieger, M., & Keck, W. (1999) Use
of genomics to identify bacterial undecaprenyl pyrophosphate synthetase: cloning,
expression and characterization of the essential uppS gene. J. Bacteriol. 181,
483-492.
[35] Guo, R. T., Kuo, C. J., Chou, C. C., Ko, T. P., Shr, H. L., Liang, P. H., & Wang, A.
H.-J. (2004) Crystal structure of octaprenyl pyrophosphate synthase from
hyperthermophilic Thermotoga maritima and mechanism of product chain length
determination. J. Biol. Chem. 279, 4903–4912.
[36] Guo, R. T., Kuo, C. J., Ko, T. P., Liang, P. H., & Wang, A. H.-J. (2004) A
molecular ruler for chain elongation catalyzed by octaprenyl pyrophosphate synthase
and its structure-based engineering to produce unprecedented long chain trans-prenyl
products. Biochemistry 43, 7678–7686.
[37] Fujihashi, M., Zhang, Y.-W., Higuchi, Y., Li, X.-Y., Koyama, T., & Miki, K.
(2001) Crystal structure of cis-prenyl chain elongating enzyme, undecaprenyl
diphosphate synthase. Proc. Natl. Acad. Sci. U. S. A. 98, 4337–4342.
[38] Ko, T.P., Chen, Y.K., Robinson, H., Tsai, P.C., Gao, Y.-G., Chen, A.P.-C., Wang,
A. H.-J., & Liang, P.H. (2001) Mechanism of product chain length determination and
the role of a flexible loop in E. coli undecaprenyl pyrophosphate synthase catalysis. J.
Biol. Chem. 276, 47474–47482.
[39] Chang, S. Y., Ko, T. P., Liang, P. H., & Wang, A. H.-J. (2003) Catalytic
mechanism revealed by the crystal structure of undecaprenyl pyrophosphate synthase
in complex with sulfate, magnesium, and triton. J. Biol. Chem. 278, 29298–29307.
[40] Chang, S. Y., Ko, T. P., Chen, A. P.-C., Wang, A. H.-J., & Liang, P. H. (2004)
Substrate binding mode and reaction mechanism of undecaprenyl pyrophosphate
synthase deduced from crystallographic studies. Protein Sci. 13, 971–978.
[41] Poulter, C. D., Argyle, J. C., & Mash, E. A. (1977) Letter: Prenyltransferase. New
evidence for an ionization-condensation-elimination mechanism with
2-fluorogeranyl pyrophosphate. J. Am. Chem. Soc. 99, 957–959.
[42] Joly, A., & Edwards, P. A. (1993) Effect of site-directed mutagenesis of
conserved aspartate and arginine residues upon farnesyl diphosphate synthase activity
J. Biol. Chem. 268, 26983–26989.
[43] Chen, A., Kroon, P. A., & Poulter, C. D. (1994) Isoprenyl diphosphate synthases:
protein sequence comparisons, a phylogenetic tree, and predictions of secondary
structure. Protein Sci. 3, 600-607.
[44] Hosfield, D. J., Zhang, Y., Dougan, D. R., Broun, A., Tari, L. W., Swanson, R. V.,
& Finn, J. (2004) Structural basis for bisphosphonate-mediated inhibition of
isoprenoid biosynthesis, J. Biol. Chem. 279, 8526–8529.
[45] Tarshis, L. C., Proteau, P. J., Kellogg, B. A., Sacchettini, J. C., & Poulter, C. D.
(1996) Regulation of product chain length by isoprenyl diphosphate synthase.
Proc. Natl. Acad. Sci. U.S.A. 93, 15018-15023.
[46] Robyt, J. (1998) In Essentials of carbohydrate chemistry Chapter 10, pp 305-318.
Springer-Verlag, New York,
[47] Allen C. M. (1985) Purification and characterization of undecaprenyl
pyrophosphate synthase. Method Enzymol. 110, 281-299.
[48] Pan, J. J., Chiou, S. T. & Liang, P. H. (2000) Product distribution and
pre-steady-state kinetic analysis of Escherichia coli undecaprenyl pyrophosphate
synthase reaction. Biochemistry 39, 10936-10942.
[49] Baba, T., & Allen, C. M. (1980) Prenyl transferases from Micrococcus luteus:
characterization of undecaprenyl pyrophosphate synthetase. Arch. Biochem. Biophys.
200, 474-484.
[50] Allen, C. M., Keenan, M. V., & Sack, J. (1976) Lactobacillus plantarum
undecaprenyl pyrophosphate synthetase: purification and reaction requirements Arch.
Biochem. Biophys. 175, 236-248.
[51] Keenan M. V., & Allen, C. M. (1974) Characterization of undecaprenyl
pyrophosphate synthetase from Lactobacillus plantarum. Arch. Biochem. Biophys.
161, 375-383.
[52] Allen, C. M., & Muth, J. D. (1977) Lipid activation of undecaprenyl
pyrophosphate synthetase from Lactobacillus plantarum Biochemistry 16, 2908-2915.
[53] Keenan, M. V., & Allen, C. M. (1974) Phospholipid activation of Lactobacillus
plantarum undecaprenyl pyrophosphate synthetase Biochem. Biophys. Res. Commun.
61, 338-342.
[54] Song, L., & Poulter, C.D. 1994. Yeast farnesyl-diphosphate synthase:
site-directed mutagenesis of residues in highly conserved prenyltransferase domains I
and II. Proc. Natl. Acad. Sci. U.S.A. 91, 3044-3048.
[55] Pan, J. J., Yang, L. W. & Liang, P. H. (2000) Effect of site-directed mutagenesis
of the conserved aspartate and glutamate on E. coli undecaprenyl pyrophosphate
synthase catalysis. Biochemistry 39, 13856-13861.
[56] Kharel, Y., Zhang, Y.-W., Fujihashi, M., Miki, K. & Koyama, T. (2001)
Identification of significant residues for homoallylic substrate binding of Micrococcus
luteus B-P 26 undecaprenyl diphosphate synthase. J. Biol. Chem. 276, 28459-28464.
[57] Fujikura, K., Zhang, Y.-W., Yoshizaki, H., Nishino, T., & Koyama, T. (2000)
Significance of Asn-77 and Trp-78 in the catalytic function of undecaprenyl
diphosphate synthase of Micrococcus luteus B-P 26. J. Biochem. (Tokyo) 128,
917-922.
[58] Kinoshita, K., Sadanami, K., Kidera, A. & Go, N. (1999) Structural motif of
phosphate-binding site common to various protein superfamilies: all-against-all
structural comparison of protein-mononucleotide complexes. Protein Eng. 12, 11-14.
[59] Chen, Y. H., Chen, A. P., Chen, C. T., Wang, A. H.-J., & Liang, P. H. (2002)
Probing the conformational change of E.coli undecaprenyl pyrophosphate synthase
during catalysis using an inhibitor and trytophan mutants. J. Biol. Chem. 277,
7369–7376.
[60] Owen, D. J., Alexandrov, K., Rostkova, E., Scheidig, A. J., Goody, R. S., &
Waldmann, H. (1999) Chemo-Enzymatic Synthesis of Fluorescent Rab 7 Proteins:
Tools to Study Vesicular Trafficking in Cells. Angew. Chem. Int. Ed. 38, 509-512.
[61] Thomä, N. H., Iakovenko, A., Owen, D., Scheidig, A. S., Waldmann, H., Goody,
R. S., & Alexandrov, K. (2000) Allosteric regulation of substrate binding and product
release in geranylgeranyltransferase type II. Biochemistry 39, 12043-12052.
[62] Liu, X.-H., & Prestwich, G. D. (2001) Didehydrogeranylgeranyl (Delta Delta
GG): a fluorescent probe for protein prenylation. J. Am. Chem. Soc. 124, 20-21.
[63] Chen, A. P.-C., Chen, Y. H., Liu, H. P., Li, Y. C., Chen, C. T., & Liang, P. H.
(2002). Synthesis and application of a fluorescent substrate analogue to study ligand
interactions for undecaprenyl pyrophosphate synthase. J. Am. Chem. Soc.
124,15217-15224.
[64] Chen, A. P.-C., Chang, S. Y., Lin, Y. C., Sun, Y. S., Chen, C. T., Wang, A. H.-J.,
& Liang, P. H. (2005) Substrate and product specificities of cis-type undecaprenyl
pyrophosphate synthase. Biochem. J. 386, 169–176.
[65] Guo, R. T., Ko, T. P., Chen , A.P., Kuo, C.J., Wang, A.H., & Liang, P. H. (2005)
Crystal structures of undecaprenyl pyrophosphate synthase in complex with
magnesium, isopentenyl pyrophosphate and farnesyl thiopyrophosphate: Roles of the
metal ion and conserved residues in catalysis. J. Biol. Chem. 280, 20762-20774.
[66] Fersht, A. (1984) In Enzyme Structure and Mechanism pp 99-101, W. H.
Freeman and company, New York.
[67] Mathis, J. R., & Poulter, C. D. (1997) Yeast protein farnesyltransferase: a
pre-steady-state kinetic analysis. Biochemistry 36, 6367-6376.
[68] Segel, I. H. (1993) in Enzyme kinetics: behavior and analysis of rapid
equilibrium and steady-state enzyme systems (Wiley Classics Library Ed.) pp
100-118, John Wiley and Sons, New York.
[69] King, H. L., & Rilling, H. C. (1977) Avian liver prenyltransferase – role of metal
in substarte binding and orientation of substrate during catalysis. Biocehmistry 16,
3815-3819.
[70] Fujii, H., Koyama, T., & Ogura, K. (1982) Efficient enzymatic hydrolysis of
polyprenyl pyrophosphates. Biochim. Biophys. Acta 712, 716-718.
[71] Lakowicz, J. R.(1999) in Principles of Fluorescecne Spectroscopy (2nd ed.)
Chapter 2, pp 52-59. Kluwer, New York
[72] Bourson, J., Pouget, J., & Valeur, B. (1993) Ion-responsive fluorescent
compounds. 4. Effect of cation binding on the photophysical properties of a coumarin
linked to monoaza- and diaza-crown ethers. J. Phys. Chem. 97, 4552-4557.
[73] Eftink, M. R. (1995) Use of multiple spectroscopic methods to monitor
equilibrium unfolding of proteins. Methods Enzymol. 259, 487-512.
[74] Sreerama, N., & Woody, R. W. (1993) A self-consistent method for the analysis
of protein secondary structure from circular dichroism. Anal. Biochem. 209, 32-44.
[75] Liang, P. H. & Anderson, K. S. (1998) Kinetic reaction scheme for the
dihydrofolate reductase domain of the bifunctional thymidylate
synthase-dihydrofolate reductase from Leishmania major. Biochemistry 37,
12206-12212.
[76] Liang, P. H. & Anderson, K. S. (1998) Substrate channeling and domain-domain
interactions in bifunctional thymidylate synthase-dihydrofolate reductase.
Biochemistry 37, 12195-12205.
[77] Reichardt, C. (1994) Solvatochromic Dyes as Solvent Polarity Indicators. Chem.
Rev. 94, 2319-2358.
[78] Kosower, E. M., & Mohammad, M. (1971) Stable free radicals. VI. Reaction
between 1-ethyl-4-carbomethoxypyridinyl radical and 4-nitrobenzyl halides. J. Am.
Chem. Soc., 93, 2713-2719.
[79] Kamlet, M. J., Abboud, J.-L. M., & Taft, R. W. (1977) Regarding a generalized
scale of solvent polarities. J. Am. Chem. Soc. 99, 8325-8327.
[80] da Costa, B. M. T., Keasling, J. D., & Cornish, K. (2005) Regulation of Rubber
Biosynthetic Rate and Molecular Weight in Hevea brasiliensis by Metal Cofactor.
Biomacromolecules, 6, 279–289.
[81] Haugland, R.P. (2002) Handbook of Fluorescent Probes and Research Product,
9th ed; Molecular Probes Inc.
[82] Hiratsuka, T. (1982) New fluorescent analogs of cAMP and cGMP available as
substrates for cyclic nucleotide phosphodiesterase. J. Biol. Chem. 257, 13354-13358.
[83] Turek, T. C., Gaon, I., Gamache, D., & Distefano, M. D. (1997) Synthesis and
evaluation of benzophenone-based photoaffinity labeling analogs of prenyl
pyrophosphates containing stable amide linkages. Bioorg. Med. Chem. Lett. 7,
2125-2130.
[84] Bukhtiyarov, Y. E., Shabalin, Y. A., & Kulaev, I. S. (1993) Solubilization and
characterization of dehydrodolichyl diphosphate synthase from the yeast
Saccharomyces carlsbergensis. J. Biochem. 113, 721-728.
[85] Chang, S. Y., Chen, Y. K., Wang, A. H.-J., & Liang, P. H. (2003) Identification of
the active conformation and the importance of length of the flexible loop 72-83 in
regulating the conformational change of undecaprenyl pyrophosphate synthase.
Biochemistry 42, 14452-14459.
[86] Wang, C. W., Oh, M. K., & Liao, J. C. (1999) Engineered isoprenoid pathway
enhances astaxanthin production in Escherichia coli. Biotechnol Bioeng. 62, 235-241.
[87] Huang, Q., Roessner, C. A., Croteau, R., & Scott, A. I. (2001) Engineering
Escherichia coli for the synthesis of taxadiene, a key intermediate in the biosynthesis
of taxol. Bioorg. Med. Chem. 9, 2237-2242.
[88] Ravanello, M. P., Ke, D., Alvarez, J., Huang, B., & Shewmaker, C. K. (2003)
Coordinate expression of multiple bacterial carotenoid genes in canola leading to
altered carotenoid production. Metab. Eng. 5, 255-263.
[89] Math, S. K., Hearst, J. E., & Poulter, C. D. (1992) The crtE gene in Erwinia
herbicola encodes geranylgeranyl diphosphate synthase. Proc. Natl. Acad. Sci. 89,
6761-6764.
[90] Asawatreratanakul, K., Zhang, Y. W., Wititsuwannakul, D., Wititsuwannakul, R.,
Takahashi, S., Rattanapittayaporn, A., & Koyama, T. (2003) Molecular cloning,
expression and characterization of cDNA encoding cis-prenyltransferases from Hevea
brasiliensis. A key factor participating in natural rubber biosynthesis. Eur. J. Biochem.
270, 4671-4680.
[91] Dursina, B., Thomä, N. H., Sidorovitch, V., Niculae, A., Iakovenko, A., Rak, A.,
Albert, S., Ceacareanu, A., Kölling, R., Herrmann, C., Goody, R. S., & Alexandrov,
K.(2002) Interaction of yeast Rab geranylgeranyl transferase with its protein and lipid
substrates. Biochemistry 41, 6805-6816.
[92] Waluk, J. (2002) Conformational Aspects of Intra- and Intermolecular Excited
State Proton Transfer; Conformational Analysis of Molecules in Excited States" (J.
Waluk, Ed.); Wiley-VCH.
[93] In some ESIPT molecules, the lowest excited singlet state is in an n  *
configuration, in which ESIPT is prohibited. For recent review, see Scheiner, S. (2000)
J. Phys. Chem. 104, 5898.
[94] Lukeman, M., & Wan, P. (2002) A New Type of Excited-State Intramolecular
Proton Transfer: Proton Transfer from Phenol OH to a Carbon Atom of an Aromatic
Ring Observed for 2-Phenylphenol. J. Am. Chem. Soc. 124, 9458-9464.
[95] McMorrow, D., & Kasha, M. (1984) Intramolecular excited-state proton transfer
in 3-hydroxyflavone. Hydrogen-bonding solvent perturbations J. Phys. Chem. 88,
2235-2245.
[96] Brucker, G. A., & Kelley, D. F. (1987) Proton transfer in matrix-isolated
3-hydroxyflavone and 3-hydroxyflavone complexe. J. Phys. Chem. 91, 2856-2861.
[97] Brucker, G. A., & Kelley, D. F. (1987) Spectroscopy and proton transfer of
matrix-isolated hydrogen-bonding 3-hydroxychromone complexes. J. Phys. Chem. 91,
2862-2866.
[98] Strandjord, A. J. G., & Barbara, P. F. (1985) The proton-transfer kinetics of
3-hydroxyflavone: solvent effects. J. Phys. Chem. 89, 2355-2361.
[99] Brucker, G. A., Kelley, D. F., & Swinney, T. C. (1991) Proton-transfer and
solvent polarization dynamics in 3-hydroxyflavone. J. Phys. Chem. 95, 3190-3195.
[100] Kraulis, P. J. (1991) MOLSCRIPT: a program to produce both detailed and
schematic plots of protein structures. J. Appl. Cryst. 24, 946-950.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top