梁博煌 教授臺灣大學：生化科學研究所陳姵君Pei-Chun, Chen AnnieChen AnniePei-Chun2007-11-262018-07-062007-11-262018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/52716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) 1414122313 bytesapplication/pdfen-US十一異戊二烯焦磷酸合成酶動力學研究類異戊二烯族Undecaprenyl Pyrophosphate synthaseisoprenyl pyrophosphate synthasekinetic studyantibacterial drug targetotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/52716/1/ntu-94-F89242018-1.pdf