Lactate dehydrogenase from the hyperthermophilic archaeon Methanococcus jannaschii: overexpression, crystallization and preliminary X-ray analysis.
L(+)-Lactate dehydrogenase (LDH) is a key enzyme in anaerobic metabolism which converts pyruvate to lactate. LDH from the hyperthermophilic archaebacterium Methanococcus jannaschii has been overexpressed in Escherichia coli and crystallized in two crystal forms at 297 K using 2-methyl-2,4-pentanediol as precipitant. Type I crystals grew rapidly and diffracted to at least 2.8 A Bragg spacing upon exposure to Cu Kalpha X-rays. X-ray diffraction data to 2.9 A have been collected from a native crystal. The type I crystal is tetragonal, belonging to the space group P4(2)2(1)2, with unit-cell parameters a = b = 99.74, c = 170.00 A. The asymmetric unit contains two LDH subunits, with a corresponding crystal volume per protein mass (V(m)) of 3.05 A(3) Da(-1) and a solvent content of 59.7%. Type II crystals, which grew more slowly, diffracted to at least 1.8 A Bragg spacing upon exposure to Cu Kalpha X-rays. X-ray diffraction data to 1.9 A have been collected from a native crystal. The type II crystal is orthorhombic, belonging to the space group P2(1)2(1)2, with unit-cell parameters a = 47.65, b = 125.10, c = 58.08 A. The asymmetric unit contains a single LDH subunit, with a corresponding crystal volume per protein mass (V(m)) of 2.50 A(3) Da(-1) and a solvent content of 50.8%. Therefore, the type II crystal is more suitable for high-resolution structure determination than the type I crystal.
Lee
B I
BI
Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151--742, South Korea.
Chang
C
C
Cho
S J
SJ
Han
G W
GW
Yu
Y G
YG
Eom
S H
SH
Suh
S W
SW
D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D019766:Genes, Archaeal; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D017017:Methanococcus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D007770:L-Lactate Dehydrogenase