Expression, purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase from Bacillus anthracis in the presence of pyruvate.
Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the lysine-biosynthesis pathway in bacteria, plants and some fungi. In this study, the expression of DHDPS from Bacillus anthracis (Ba-DHDPS) and the purification of the recombinant enzyme in the absence and presence of the substrate pyruvate are described. It is shown that DHDPS from B. anthracis purified in the presence of pyruvate yields greater amounts of recombinant enzyme with more than 20-fold greater specific activity compared with the enzyme purified in the absence of substrate. It was therefore sought to crystallize Ba-DHDPS in the presence of the substrate. Pyruvate was soaked into crystals of Ba-DHDPS prepared in 0.2 M sodium fluoride, 20%(w/v) PEG 3350 and 0.1 M bis-tris propane pH 8.0. Preliminary X-ray diffraction data of the recombinant enzyme soaked with pyruvate at a resolution of 2.15 A are presented. The pending crystal structure of the pyruvate-bound form of Ba-DHDPS will provide insight into the function and stability of this essential bacterial enzyme.
Voss
Jarrod E
JE
Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia.
Scally
Stephen W
SW
Taylor
Nicole L
NL
Dogovski
Con
C
Alderton
Malcolm R
MR
Hutton
Craig A
CA
Gerrard
Juliet A
JA
Parker
Michael W
MW
Dobson
Renwick C J
RC
Perugini
Matthew A
MA
D001408:Bacillus anthracis / Q000201:enzymology* / Q000235:genetics; D003460:Crystallization; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D006836:Hydro-Lyases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D019289:Pyruvic Acid / Q000737:chemistry*; D013379:Substrate Specificity / Q000235:genetics; D014961:X-Ray Diffraction / Q000379:methods
D007527:Isoenzymes; D019289:Pyruvic Acid; D006836:Hydro-Lyases; C020040:4-hydroxy-tetrahydrodipicolinate synthase