Mechanistic and Kinetic Studies of E. coli Undecaprenyl Pyrophosphate Synthase : Application in Antibacterial Drug Discovery
Date Issued
2005
Date
2005
Author(s)
Pei-Chun, Chen Annie
DOI
en-US
Abstract
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.
Subjects
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 synthase
isoprenyl pyrophosphate synthase
kinetic study
antibacterial drug target
Type
other
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