Structure determination and refinment of Bacillus stearothermophilus lactate dehydrogenase

Piontek, Klaus ; Chakrabarti, Pinakpani ; Schar, Hans-Peter ; Rossmann, Michael G. ; Zuber, Herbert (1990) Structure determination and refinment of Bacillus stearothermophilus lactate dehydrogenase Proteins: Structure, Function, and Bioinformatics, 7 (1). pp. 74-92. ISSN 0887-3585

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Structures have been determined of Bacillus stearothermophilus "apo"and holo lactate dehydrogenase. The holo-enzyme had been co-crystallized with the activator fructose 1,6-biosphosphate. The "apo"lactate dehydrogenase structure was solved by use of the known apo-M4 dogfish lactate dehydrogenase molecule as a starting model. Phases were refined and extended from 4 Å resolution by means of the noncrystallographic molecular 222 symmetry. The R-factor was reduced to 28.7%, using 2.8 Å resolution data, in a restrained least-squares refnement in which the molecula rsymmetry was imposed as a constraint. A low occupancy of coenzyme was found in each of the four subunits of the "apo"enzyme. Further refinement proceeded with the isomorphous holo-enzyume from Bacillus Stearothermophilus. After removing the noncrystallographic constraints, the R-factor dropped from 30.3% to a final value of 26.0% with a 0.019 Å and 1.7° r.m.s. deviation from idealized bond length and angles, respectively. Two sulfate ions per subunit were included in the final model of the "apo"-from-one at the substrate binding site and one close to the molecular P -axis near the location of the fructose 1,6-bisphosphate activator. The final model of the holo-enzyme incorporated two sulfate ions per subunit, one at the substrate binding site and another close to the R-axis. One nicotinamide adenine dinucleotide coenzyme molecule per subunit and two fructose 1,6-bisphosphate molecules per tetramer were also included. The phosphate positions of fructose 1,6-bisphosphate are close to the sulfate ion near the P-axis in the "apo"model. This structure represents the first reported refined model of an allosteric activated lactate dehydrogenase. The structure of the activated holo-enzyme showed far greater similarity to the ternary complex of dogfish M4 lactate dehydrogenase with incotinamide adenine dinucleotide and oxamate than to apo-M4 dogfish lactate dehydrogenase. The conformations of nicotinamide adenine dinucleotide and fructose, 1,6-bisphosphate were also analyzed.

Item Type:Article
Source:Copyright of this article belongs to John Wiley and Sons, Inc.
Keywords:Thermostability; Bacterial Lactate Dehydrogenase; Allosteric Regulations; Crystallography; Molecular Replacement; Coenzyme Binding
ID Code:21441
Deposited On:22 Nov 2010 11:25
Last Modified:01 Feb 2011 06:36

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