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

Mechanistic and Kinetic Studies of E. coli Undecaprenyl Pyrophosphate Synthase : Application in Antibacterial Drug Discovery

陳姵君(Chen Annie Pei-Chun)

指導教授 ： 梁博煌 教授

國立臺灣大學/生命科學院/生化科學研究所/博士(2005年)

https://doi.org/10.6342/NTU.2005.02254

摘要

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。

關鍵字

十一異戊二烯焦磷酸合成酶動力學研究 ； 類異戊二烯族

並列摘要

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.

並列關鍵字

Undecaprenyl Pyrophosphate synthase ； isoprenyl pyrophosphate synthase ； kinetic study ； antibacterial drug target

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