Archaeal ADP-dependent phosphofructokinase: expression, purification, crystallization and preliminary crystallographic analysis.
Thermococcus litoralis uses a modified Embden-Meyerhof pathway. Its phosphofructokinase (TLPFK) catalyses the phosphorylation of fructose-6-phosphate by ADP, but not by ATP, in the presence of Mg(2+), yielding fructose-1,6-bisphosphate. The gene encoding TLPFK was cloned and overexpressed in Escherichia coli. Recombinant TLPFK consists of a dimer with a 52 kDa subunit. The native crystals belong to space group P4(1)2(1)2, with unit-cell parameters a = b = 85.41, c = 163.93 A, and diffract to beyond 2.6 A resolution. The TLPFK structure was preliminarily analyzed by means of multiple isomorphous replacement with four heavy-atom derivatives.
Jeong
Jong-Jin
JJ
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Fushinobu
Shinya
S
Ito
Sohei
S
Shoun
Hirofumi
H
Wakagi
Takayoshi
T
D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C442129:ADP D-fructose-6-phosphate 1-phosphotransferase