Structure determination and refinement of Bacillus stearothermophilus lactate dehydrogenase. Export

@article{citeulike:4450749, abstract = {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-bisphosphate. 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 A to 3 A resolution by means of the noncrystallographic molecular 222 symmetry. The R-factor was reduced to 28.7\%, using 2.8 A resolution data, in a restrained least-squares refinement in which the molecular symmetry 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-enzyme 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 A and 1.7 degrees r.m.s. deviation from idealized bond lengths and angles, respectively. Two sulfate ions per subunit were included in the final model of the "apo"-form--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 nicotinamide 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.}, author = {Piontek, K. and Chakrabarti, P. and Sch\"{a}r, H. P. and Rossmann, M. G. and Zuber, H.}, citeulike-article-id = {4450749}, citeulike-linkout-0 = {http://dx.doi.org/10.1002/prot.340070108}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/2330370}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=2330370}, doi = {10.1002/prot.340070108}, issn = {0887-3585}, journal = {Proteins}, keywords = {ldh}, number = {1}, pages = {74--92}, posted-at = {2009-05-02 02:47:26}, priority = {2}, title = {Structure determination and refinement of Bacillus stearothermophilus lactate dehydrogenase.}, url = {http://dx.doi.org/10.1002/prot.340070108}, volume = {7}, year = {1990} }

TY - JOUR ID - citeulike:4450749 L3 - citeulike-article-id:4450749 N2 - 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-bisphosphate. 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 A to 3 A resolution by means of the noncrystallographic molecular 222 symmetry. The R-factor was reduced to 28.7%, using 2.8 A resolution data, in a restrained least-squares refinement in which the molecular symmetry 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-enzyme 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 A and 1.7 degrees r.m.s. deviation from idealized bond lengths and angles, respectively. Two sulfate ions per subunit were included in the final model of the "apo"-form--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 nicotinamide 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. IS - 1 JF - Proteins SN - 0887-3585 EP - 92 TI - Structure determination and refinement of Bacillus stearothermophilus lactate dehydrogenase. VL - 7 SP - 74 KW - ldh AU - Piontek, K AU - Chakrabarti, P AU - Schär, HP AU - Rossmann, MG AU - Zuber, H PY - 1990/// UR - http://dx.doi.org/10.1002/prot.340070108 ER -