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PDBsum entry 1v0j
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References listed in PDB file
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Key reference
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Title
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Crystal structures of mycobacteria tuberculosis and klebsiella pneumoniae udp-Galactopyranose mutase in the oxidised state and klebsiella pneumoniae udp-Galactopyranose mutase in the (active) reduced state.
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Authors
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K.Beis,
V.Srikannathasan,
H.Liu,
S.W.Fullerton,
V.A.Bamford,
D.A.Sanders,
C.Whitfield,
M.R.Mcneil,
J.H.Naismith.
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Ref.
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J Mol Biol, 2005,
348,
971-982.
[DOI no: ]
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PubMed id
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Abstract
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Uridine diphosphogalactofuranose (UDP-Galf) is the precursor of the
d-galactofuranose sugar found in bacterial and parasitic cell walls, including
those of many pathogens. UDP-Galf is made from UDP-galactopyranose by the enzyme
UDP-galactopyranose mutase. The enzyme requires the reduced FADH- co-factor for
activity. The structure of the Mycobacterium tuberculosis mutase with FAD has
been determined to 2.25 A. The structures of Klebsiella pneumoniae mutase with
FAD and with FADH- bound have been determined to 2.2 A and 2.35 A resolution,
respectively. This is the first report of the FADH(-)-containing structure. Two
flavin-dependent mechanisms for the enzyme have been proposed, one, which
involves a covalent adduct being formed at the flavin and the other based on
electron transfer. Using our structural data, we have examined the two
mechanisms. The electron transfer mechanism is consistent with the structural
data, not surprisingly, since it makes fewer demands on the precise positioning
of atoms. A model based on a covalent adduct FAD requires repositioning of the
enzyme active site and would appear to require the isoalloxazine ring of FADH-
to buckle in a particular way. However, the FADH- structure reveals that the
isoalloxazine ring buckles in the opposite sense, this apparently requires the
covalent adduct to trigger profound conformational changes in the protein or to
buckle the FADH- opposite to that seen in the apo structure.
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Figure 1.
Figure 1. The mutase enzyme function and structure. (a) The
chemical reaction catalysed by the mutase enzyme. The co-factor
FAD and FADH - are shown. Ring positions numbers are referred to
in the text. (b) The bicyclic mechanism, which does not require
direct involvement of the FAD.8 (c) The redox mechanism, in
which one electron is transferred to the substrate.11 The
electron is transferred from FADH - to create a radical that
then re-arranges to give product. (d) The covalent intermediate
mechanism, in this N5 attacks C1 in a nucleophilic manner. The
covalent intermediate then re-arranges to give product. The
presence of the covalent intermediate was detected by mass
specotrometry.12
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Figure 3.
Figure 3. Stereo images of models of substrate with mutase.
(a) The initial complex between UDP-galactose of the active
reduced form of the K. pneumoniae enzyme. This complex is
predicted to occur in a mechanism involving electron transfer or
a covalent intermediate. No significant re-arrangements are
required to accommodate the substrate. The structurally diverse
loop 5 is shown in pink. (b) A model of the covalent adduct with
the re-face buckle of isoalloxazine ring. The re-face buckled
isoalloxazine ring is taken from a thioredoxin structure.17 This
model allows interactions with key conserved residues. The model
would require conformation changes in side-chain positions only
to avoid steric clashes. (c) The covalent adduct based on the
experimental K. pneumoniae FADH - structure. The sugar is
interpenetrating with the protein structure. Either FADH -
adopts a different buckle in the presence of substrate or the
protein undergoes a profound conformation change. His63 has been
omitted for clarity and Pro59 has been added to this Figure.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2005,
348,
971-982)
copyright 2005.
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Secondary reference #1
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Title
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Udp-Galactopyranose mutase has a novel structure and mechanism.
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Authors
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D.A.Sanders,
A.G.Staines,
S.A.Mcmahon,
M.R.Mcneil,
C.Whitfield,
J.H.Naismith.
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Ref.
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Nat Struct Biol, 2001,
8,
858-863.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1. Role of UDP-galactopyranose mutase in the biosynthesis
of the cell wall. a, Cell wall of M. tuberculosis showing key
role of galactofuranose. b, Reaction catalyzed by
UDP-galactopyranose mutase, the interconversion of UDP-Galp
(left) to UDP-Galf (right). Note that there is no net loss or
gain of electrons during the contraction of the ring.
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Figure 3.
Figure 3. Putative active site of UDP-galactopyranose mutase.
a, Electrostatic plot of the surface of the protein. The
structure is orientated as Fig. 2a. The positive patch on the
surface is identified as the binding site for the negatively
charged sugar nucleotide substrate. Figure generated with
GRASP36. b, Close-up stereo view of the substrate binding cleft.
The orientation is as Fig. 2a. The absolutely conserved residues
and the flavin are shown in stick format. The color scheme is as
Fig. 2b.
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The above figures are
reproduced from the cited reference
with permission from Macmillan Publishers Ltd
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