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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 substrate specificity in malate dehydrogenases: crystal structure of a ternary complex of porcine cytoplasmic malate dehydrogenase, Alpha-Ketomalonate and tetrahydonad.
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Authors
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A.D.Chapman,
A.Cortés,
T.R.Dafforn,
A.R.Clarke,
R.L.Brady.
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Ref.
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J Mol Biol, 1999,
285,
703-712.
[DOI no: ]
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PubMed id
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Abstract
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The structural basis for the extreme discrimination achieved by malate
dehydrogenases between a variety of closely related substrates encountered
within the cell has been difficult to assess because of the lack of an
appropriate catalytically competent structure of the enzyme. Here, we have
determined the crystal structure of a ternary complex of porcine cytoplasmic
malate dehydrogenase with the alternative substrate alpha-ketomalonate and the
coenzyme analogue 1,4,5,6-tetrahydronicotinamide. Both subunits of the dimeric
porcine heart, and from the prokaryotes Escherichia coli and Thermus flavus.
However, large changes are noted around the active site, where a mobile loop now
closes to bring key residues into contact with the substrate. This observation
substantiates a postulated mechanism in which the enzyme achieves high levels of
substrate discrimination through charge balancing in the active site. As the
activated cofactor/substrate complex has a net negative charge, a positive
counter-charge is provided by a conserved arginine in the active site loop. The
enzyme must, however, also discriminate against smaller substrates, such as
pyruvate. The structure shows in the closed (loop down) catalytically competent
complex two arginine residues (91 and 97) are driven into close proximity.
Without the complimentary, negative charge of the substrate side-chain of
oxaloacetate or alpha-ketomalonate, charge repulsion would resist formation
production of this catalytically productive conformation, hence minimising the
effectiveness of pyruvate as a substrate. By this mechanism, malate
dehydrogenase uses charge balancing to achieve fivefold orders of magnitude in
discrimination between potential substrates.
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Figure 7.
Figure 7. The position of the α-ketomalonate substrate
within the active site. The electron density was generated by
calculating 2|F[obs]|−|F[calc]| difference maps contoured at
1.3σ.
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Figure 8.
Figure 8. The hydrogen-bonding interactions of
α-ketomalonate with the key active site residues. This Figure
was generated using the program LIGPLOT [Wallace et al 1995].
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(1999,
285,
703-712)
copyright 1999.
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Secondary reference #1
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Title
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Refined crystal structure of cytoplasmic malate dehydrogenase at 2.5-A resolution.
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Authors
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J.J.Birktoft,
G.Rhodes,
L.J.Banaszak.
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Ref.
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Biochemistry, 1989,
28,
6065-6081.
[DOI no: ]
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PubMed id
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Secondary reference #2
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Title
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Structure of porcine heart cytoplasmic malate dehydrogenase: combining X-Ray diffraction and chemical sequence data in structural studies.
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Authors
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J.J.Birktoft,
R.A.Bradshaw,
L.J.Banaszak.
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Ref.
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Biochemistry, 1987,
26,
2722-2734.
[DOI no: ]
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PubMed id
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Secondary reference #3
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Title
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The presence of a histidine-Aspartic acid pair in the active site of 2-Hydroxyacid dehydrogenases. X-Ray refinement of cytoplasmic malate dehydrogenase.
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Authors
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J.J.Birktoft,
L.J.Banaszak.
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Ref.
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J Biol Chem, 1983,
258,
472-482.
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PubMed id
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Secondary reference #4
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Title
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The interactions of NAD/nadh with 2-Hydroxy acid dehydrogenases
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Authors
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J.J.Birktoft,
R.T.Fernley,
R.A.Bradshaw,
L.J.Banaszak.
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Ref.
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molecular structure and ...
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Secondary reference #5
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Title
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Amino acid sequence homology among the 2-Hydroxy acid dehydrogenases: mitochondrial and cytoplasmic malate dehydrogenases form a homologous system with lactate dehydrogenase.
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Authors
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J.J.Birktoft,
R.T.Fernley,
R.A.Bradshaw,
L.J.Banaszak.
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Ref.
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Proc Natl Acad Sci U S A, 1982,
79,
6166-6170.
[DOI no: ]
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PubMed id
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Secondary reference #6
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Title
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Nicotinamide adenine dinucleotide and the active site of cytoplasmic malate dehydrogenase
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Authors
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L.J.Banaszak,
L.E.Webb.
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Ref.
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structure and conformation ...
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Secondary reference #7
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Title
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Conformation of nicotinamide adenine dinucleotide bound to cytoplasmic malate dehydrogenase.
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Authors
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L.E.Webb,
E.J.Hill,
L.J.Banaszak.
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Ref.
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Biochemistry, 1973,
12,
5101-5109.
[DOI no: ]
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PubMed id
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Secondary reference #8
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Title
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Polypeptide conformation of cytoplasmic malate dehydrogenase from an electron density map at 3.0 angstrom resolution.
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Authors
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E.Hill,
D.Tsernoglou,
L.Webb,
L.J.Banaszak.
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Ref.
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J Mol Biol, 1972,
72,
577-589.
[DOI no: ]
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PubMed id
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Figure 1.
FIG. 1. (a) Sohemetio di8gmm of polypeptide chain folding in the s-MDH subunit. The subunit
is divided into two parts for oonvenienoe of illu&retion with the dotted line indicating the oon-
t&&y of the polypeptide ohain. The nomenolature is described in the text. (b) Stereo drawinga
of the a-carbon beckbone of s-YDH (subunit I). The view is suoh that the to parts shown in
(8) are appamnt. The oonneotig segment oorresponding to the dotted line in Fig. l(8) is indioeted
by 8n arrow. The symbola C and N mark the oarboxyl and amino terming respeotively.
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Figure 4.
IG. 4. See legend to Fig. 2. Stereo vew of the polypepide ohain of e-MDH from a2B to alcf.
The orient&ion is approxim&ely the same aa thet in Fig. l(b); a slight rotation wea ne0esaer.y
to optimize the viewing angle. The first anti-parallel ribbon, consisting of the atrends Pa and fiH,
ia prominent eetwe.
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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Secondary reference #9
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Title
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The identification of an asymmetric complex of nicotinamide adenine dinucleotid and pig heart cytoplasmic malate dehydrogenase.
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Authors
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B.E.Glatthaar,
L.J.Banaszak,
R.A.Bradshaw.
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Ref.
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Biochem Biophys Res Commun, 1972,
46,
757-765.
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PubMed id
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Secondary reference #10
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Title
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Cytoplasmic malate dehydrogenase--Heavy atom derivatives and low resolution structure.
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Authors
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D.Tsernoglou,
E.Hill,
L.J.Banaszak.
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Ref.
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J Mol Biol, 1972,
69,
75-87.
[DOI no: ]
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PubMed id
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Figure 3.
FIG. 3. Three-dimensions1 difference Patterson map of PHMS derivative. The reflexions included
in the calculations correpond to 5.5 L% spacings. (a) Section z =0, (b) section z = l/2, (c) section
y= l/2. The numbers 1, 2, 3 indicated on esoh section mark the location of the vectors resulting
rom the symmetry related positions of esch heavy atom site (see text).
6
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Figure 6.
FIG. 6. Sections f three-dimensional difference Fourier maps (6.6 d resolution) of PtClf - 8nd
platinum ethylene diamine dichloride derivatves. The phases used for the calculations are described
in the text. (a) PtCli- derivative, (b) platinum ethylene diamine dichloride drivative. The section
shown contains one of the major platinum binding sites. This is indicated by 1. In addition, this
section ould contain one of the PHMS binding sites used for the phase calculation. It is indicated
by 2.
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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Secondary reference #11
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Title
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Structural studies on heart muscle malate dehydrogenases.
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Authors
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D.Tsernoglou,
E.Hill,
L.J.Banaszak.
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Ref.
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Cold Spring Harb Symp Quant Biol, 1972,
36,
171-178.
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PubMed id
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