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PDBsum entry 2pyh
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References listed in PDB file
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Key reference
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Title
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Structural and mutational characterization of the catalytic a-Module of the mannuronan c-5-Epimerase alge4 from azotobacter vinelandii.
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Authors
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H.J.Rozeboom,
T.M.Bjerkan,
K.H.Kalk,
H.Ertesvåg,
S.Holtan,
F.L.Aachmann,
S.Valla,
B.W.Dijkstra.
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Ref.
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J Biol Chem, 2008,
283,
23819-23828.
[DOI no: ]
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PubMed id
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Abstract
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Alginate is a family of linear copolymers of (1-->4)-linked beta-d-mannuronic
acid and its C-5 epimer alpha-l-guluronic acid. The polymer is first produced as
polymannuronic acid and the guluronic acid residues are then introduced at the
polymer level by mannuronan C-5-epimerases. The structure of the catalytic
A-module of the Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 has been
determined by x-ray crystallography at 2.1-A resolution. AlgE4A folds into a
right-handed parallel beta-helix structure originally found in pectate lyase C
and subsequently in several polysaccharide lyases and hydrolases. The beta-helix
is composed of four parallel beta-sheets, comprising 12 complete turns, and has
an amphipathic alpha-helix near the N terminus. The catalytic site is positioned
in a positively charged cleft formed by loops extending from the surface
encompassing Asp(152), an amino acid previously shown to be important for the
reaction. Site-directed mutagenesis further implicates Tyr(149), His(154), and
Asp(178) as being essential for activity. Tyr(149) probably acts as the proton
acceptor, whereas His(154) is the proton donor in the epimerization reaction.
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Figure 5.
FIGURE 5. Superimposition of the active site residues of
ALY-1 in green (His^119, Gln^117, Arg^72, and Tyr^195), A1-III
(His^192, Asn^191, Arg^239, Tyr^246, and trisaccharide) in
lilac, and AlgE4A (His^154, Asp^152, Lys^117, and Tyr^149 and
trisaccharide) in CPK colors. The image was constructed in PyMOL
(52).
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Figure 7.
FIGURE 7. Proposed catalytic mechanism of AlgE4. A and B,
the alginate polymer enters the catalytic site. B and C, the
carboxylate moiety of the mannuronic acid in subsite +1 is
protonated, enabling it to form a hydrogen bond with Asp^152
(and/or 178), which stabilizes the substrate-enzyme complex. C,
upon deprotonation of Tyr^149 (via Arg^195) the alkoxide ion
group extracts H-5 from the re-face of the mannuronic acid in
subsite +1. C and D, a double bond is formed, which makes the
conformation of the +1 mannuronic acid partially planar. D, the
protonated His^154 performs a nucleophilic attack on the C-5
atom of the +1 sugar from the si-face with the concomitant flip
of the +1 sugar ring into the ^1C[4] chair conformation of
guluronic acid. D and E, the carboxylic acid moiety on sugar +1
is deprotonated. E and F, the epimerized sugar leaves the active
site and His^154 is protonated again. F, the epimerase is ready
to perform a new reaction.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2008,
283,
23819-23828)
copyright 2008.
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