Cloning, expression, purification and crystallization of dihydrodipicolinate synthase from the psychrophile Shewanella benthica.
Dihydrodipicolinate synthase (DHDPS) is an oligomeric enzyme that catalyzes the first committed step of the lysine-biosynthesis pathway in plants and bacteria, which yields essential building blocks for cell-wall and protein synthesis. DHDPS is therefore of interest to drug-discovery research as well as to studies that probe the importance of quaternary structure to protein function, stability and dynamics. Accordingly, DHDPS from the psychrophilic (cold-dwelling) organism Shewanella benthica (Sb-DHDPS) was cloned, expressed, purified and crystallized. The best crystals of Sb-DHDPS were grown in 200 mM ammonium sulfate, 100 mM bis-tris pH 5.0-6.0, 23-26%(w/v) PEG 3350, 0.02%(w/v) sodium azide and diffracted to beyond 2.5 Å resolution. Processing of diffraction data to 2.5 Å resolution resulted in a unit cell with space group P2(1)2(1)2(1) and dimensions a = 73.1, b = 84.0, c = 143.7 Å. These studies of the first DHDPS enzyme to be characterized from a bacterial psychrophile will provide insight into the molecular evolution of enzyme structure and dynamics.
Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia.
Jacinta M
JM
Wubben
Con
C
Dogovski
Renwick C J
RC
Dobson
Rachel
R
Codd
Juliet A
JA
Gerrard
Michael W
MW
Parker
Matthew A
MA
Perugini
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D015870:Gene Expression; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D020592:Shewanella / Q000201:enzymology*
D006836:Hydro-Lyases; C020040:4-hydroxy-tetrahydrodipicolinate synthase