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PDBsum entry 1uxt
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Oxidoreductase
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PDB id
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1uxt
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Contents |
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* Residue conservation analysis
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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 basis of allosteric regulation and substrate specificity of the non-Phosphorylating glyceraldehyde 3-Phosphate dehydrogenase from thermoproteus tenax.
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Authors
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E.Lorentzen,
R.Hensel,
T.Knura,
H.Ahmed,
E.Pohl.
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Ref.
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J Mol Biol, 2004,
341,
815-828.
[DOI no: ]
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PubMed id
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Abstract
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The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) of the
hyperthermophilic Archaeum Thermoproteus tenax is a member of the superfamily of
aldehyde dehydrogenases (ALDH). GAPN catalyses the irreversible oxidation of
glyceraldehyde 3-phosphate (GAP) to 3-phosphoglycerate in the modified
glycolytic pathway of this organism. In contrast to other members of the ALDH
superfamily, GAPN from T.tenax (Tt-GAPN) is regulated by a number of
intermediates and metabolites. In the NAD-dependent oxidation of GAP, glucose
1-phosphate, fructose 6-phosphate, AMP and ADP increase the affinity for the
cosubstrate, whereas ATP, NADP, NADPH and NADH decrease it leaving, however, the
catalytic rate virtually unaltered. As we show here, the enzyme also uses NADP
as a cosubstrate, displaying, however, unusual discontinuous saturation kinetics
indicating different cosubstrate affinities and/or reactivities of the four
active sites of the protein tetramer caused by cooperative effects. Furthermore,
in the NADP-dependent reaction the presence of activators decreases the overall
S0.5 and increases Vmax by a factor of 3. To explore the structural basis for
the different effects of both pyridine nucleotides we solved the crystal
structure of Tt-GAPN in complex with NAD at 2.2 A resolution and compared it to
the binary Tt-GAPN-NADPH structure. Although both pyridine nucleotides show a
similar binding mode, NADPH appears to be more tightly bound to the protein via
the 2' phosphate moiety. Moreover, we present four co-crystal structures with
the activating molecules glucose 1-phosphate, fructose 6-phosphate, AMP and ADP
determined at resolutions ranging from 2.3 A to 2.6 A. These crystal structures
reveal a common regulatory site able to accommodate the different activators. A
phosphate-binding pocket serves as an anchor point ensuring similar binding
geometry. The observed conformational changes upon activator binding are
discussed in terms of allosteric regulation. Furthermore, we present a crystal
structure of Tt-GAPN in complex with the substrate D-GAP at 2.3 A resolution,
which allows us to analyse the structural basis for substrate binding, the
mechanism of catalysis as well as the stereoselectivity of the enzymatic
reaction.
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Figure 2.
Figure 2. (a) NADP saturation of Tt-GAPN in the absence of
activator. The insert shows the concentration range of 0-0.5 mM
NADP. (b) NADP saturation of Tt-GAPN in the absence (sB) and
presence of 50 µM G1P (cD-).
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Figure 5.
Figure 5. Hydrogen-bonding network involving the C-terminal
carboxyl of a symmetry equivalent monomer, the phosphate moiety
of an activator, the carbonyl of Arg72 and a well ordered water
molecule. The binding of activators leads to a considerable
fixation of the otherwise partially disordered C terminus of the
protein.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2004,
341,
815-828)
copyright 2004.
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