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Crystallization and preliminary X-ray diffraction studies of two mutants of lactate dehydrogenase from Bacillus stearothermophilus

✍ Scribed by Kui Huang; R. Kodandapani; Helmut Kallwass; James K. Hogan; Wendy Parris; James D. Friesen; Marvin Gold; J. Bryan Jones; Dr. Michael N. G. James

Publisher
John Wiley and Sons
Year
1992
Tongue
English
Weight
375 KB
Volume
13
Category
Article
ISSN
0887-3585

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✦ Synopsis

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 proteincan 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 (NH~4~)~2~SO~4~in 50 mM piperazine HCI buffer. The crystals belong to space group P6~6~22 (P6~6~22) (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 Å, c = 168.6 Å for the R171W, Q102R, C97G triple mutant, and a = 98.2 Å; c = 162.1 Å for the R171Y, Q102R, C97G mutant. These crystal forms appear to contain one‐quarter of a tetramer (Mr 135,000)in the asymmetric unit and have (V~M~ values of 3.8 and 3.3 Å^3^/dalton, respectively). The R171W mutant diffracts to 2.5 Å and the R171Y mutant to approximately 3.5 Å © 1992 Wiley‐Liss, Inc.

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