title: Crystallization and preliminary X-ray diffraction studies of two mutants of lactate dehydrogenase from Bacillus stearothermophilus.
abstract: Bacillus stearothermophilus lactate dehydrogenase, one of the most thermostable bacterial enzymes known, has had its three-dimensional structure solved, the gene coding for it has been cloned, and the protein can be readily overexpressed. Two mutants of the enzyme have been prepared. In one, Arg171 was changed to Trp (R171W) and Gln102 was changed to Arg (Q102R). In the other, the mutation Q102R was maintained, but Arg171 was changed to Tyr (R171Y). In addition, an inadvertent C97G mutant was present. Both mutants have been crystallized by the hanging drop vapor diffusion method at room temperature. Bipyrimidal crystals have been obtained against (NH4)2SO4 in 50 mM piperazine HCl buffer. The crystals belong to space group P6(2)22 (P6(4)22) (whereas the native enzyme, the structure of which has been solved by Piontek et al., Proteins 7:74-92, 1990) crystallized in the space group P6(1)) with a = 102.3 A, c = 168.6 A for the R171W, Q102R, C97G triple mutant, and a = 98.2 A; c = 162.1 A for the R171Y, Q102R, C97G mutant. These crystal forms appear to contain one-quarter of a tetramer (M(r) 135,000) in the asymmetric unit and have VM values of 3.8 and 3.3 A3/dalton, respectively). The R171W mutant diffracts to 2.5 A and the R171 Y mutant to approximately 3.5 A.
authors.affiliation: Medical Research Council of Canada Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton.
authors.forename: K
authors.initials: K
authors.lastname: Huang
authors.forename: R
authors.initials: R
authors.lastname: Kodandapani
authors.forename: H
authors.initials: H
authors.lastname: Kallwass
authors.forename: J K
authors.initials: JK
authors.lastname: Hogan
authors.forename: W
authors.initials: W
authors.lastname: Parris
authors.forename: J D
authors.initials: JD
authors.lastname: Friesen
authors.forename: M
authors.initials: M
authors.lastname: Gold
authors.forename: J B
authors.initials: JB
authors.lastname: Jones
authors.forename: M N
authors.initials: MN
authors.lastname: James
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D003460:Crystallization; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D007770:L-Lactate Dehydrogenase