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The substrate binding pocket of the active site comprises large number
of loop regions two of which are provided by the adjacent subunit across the
dimer interface. In the ternary complex, FMN is almost completely buried, with
part of its accessible surface area being occluded by the substrate, EPSP. Such
an arrangement is indicative of an ordered binding of FMN followed by EPSP as
shown by previous rapid kinetic studies (Macheroux et al 1998). CS derived from S. pneumoniae contains the FMN molecule making few
specific polar interactions with protein majorly including contacts between
hydroxyl and phosphate oxygens of the ribityl chain. The flexible regions around
the FMN site majorly comprise of highly conserved residues. The FMN phosphate
is present at the dimerization interface involved in numerous contacts with
residues from the adjacent monomer. In addition there are many solvent
molecules adjacent to both the phosphate and ribityl regions of FMN. The water molecules
which are responsible for mediating interactions between FMN and the
surrounding residues are discrete and ordered. FMN contains a hydrophobic isoalloxazine
ring system that is responsible for few specific interactions with the protein.
This structure buries a significant area of hydrophobic surface by packing the re-face
against the complementary surface of the protein Maclean and Ali 2003. This arrangement
helps in lowering flavin´s reduction potential to a value comparable to the
most reducing flavodoxins Bornemann et al 2003. There is a considerable deviation
from planarity displayed by isoalloxazine ring in bound FMN. The pyrimidine
ring and the dimethyl benzene ring make an angle of approximately 10º and the re-face
of the isolloxazine ring is convex assists the substrate EPSP-binding site. (Ahn
et al 2004, Binda et al 1999, Yue et al 1999,Barber et al
1992). The FMN binding site has numerous positive charges in the proximity of
the isolloxazine ring causing an elevation in the redox potential of FMN. The positive
charges at this position can potentially stabilize negatively charged N1 in the
reduced flavin, thus keeping the electron until the substrate EPSP is bound 60.The EPSP molecule is oriented relative to the N5
position of the isoalloxazine ring for electron transfer and hydrogen atom
abstraction to and from the substrate, respectively. The EPSP molecule is
attacked above the si-face of the isoalloxazine ring in an average
distance of 3.3 Å as in the structure of CS from S. Pneumoniae. Due to
such orientation there is no accumulation of flavin radical intermediates on
the stopped-flow millisecond time scale. Therefore CS catalyzes a unique reaction in
flavoenzyme class where there is transfer of only one electron. The
localization of the FMN and EPSP in the CS has provided evidence that the
flavin is well placed to abstract a hydrogen atom from the 6-proR position
of the EPSP molecule, rather than just an electron with the proton being
accepted by an amino acid side chain. This form of flavin can donate an
electron and subsequently accept a hydrogen atom, stabilizing again (Maclean
and Ali 2003, Bornemann et al 2003).In
the structure of CS from S. pneumoniae, the EPSP molecule makes a number
of polar interactions with the protein, and few hydrophobic contacts. The site
of EPSP is hydrophilic and has an environment extensively basic with many basic
residues, mainlyarginines around of the binding site, which are extremely
conserved in many sequences, thereby showing its importance in the charge stabilization
of EPSP molecule (Maclean and Ali 2003),observed that the quaternary structure of
 S. pneumoniae has two
conformational states. One of the four monomers of the tetramer has differences
when compared with the other three. These changes occur principally in two
loops of active site comprised of highly conserved residues. The quaternary structure
shows one monomer with the active site more accessible having therefore an open
conformation, whereas the other three have closed conformations. These
differences provide vital information about the mobility of these active site
loops, which play an important mechanistic role (Maclean and Ali 2003). These
loops have important basic residues related with the catalytic mechanism of the
protein. Differences between open and closed active sites demonstrate that the side chain of one
histidine located in one of these loops has some conformational flexibility,
and interacts with the FMN in the open form but with O12 of EPSP in the closed
form. Furthermore other highly conserved residues in these loops also have
different conformations in the two states showing that these residues play
important role in the catalytic mechanism (Maclean and Ali 2003,Ahn et al 2004)
have observed also that the structure from H. pylori is surrounded by
flexible regions that are also highly conserved. (Ahn et al 2004) 60
crystallized the apo enzyme and CS complexed with FMN. The authors have
observed that FMN could cause conformational changes in these loops in CS from H.
pylori, furthermore, they have observed that the movement of these regions
results in a more apolar environment for the bound cofactor 60. This structural informationconfirms the
hypothesis, in which the CS undergoes a major change on binding oxidized flavin
and EPSP and that the apoenzym eexhibits more conformational flexibility tha

n ternary complex. Furthermore it was observed that alterations occurred
in the visible CD spectrum of the enzyme-bound FMN on binding of EPSP showing
that the environment of the flavin changes considerably(56).

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Shikimate kinase (EC 2.7.1.71) is fifth enzyme of the shikimate
pathway that catalyzes the ATP-dependent phosphorylation of shikimate to form shikimate 3-phosphate  (Herrmann, K.M.et al.,1999).
which is used by plants and bacteria to synthesize the common precursor of
aromatic amino acids and secondary metabolites. 

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