Cloning, expression, crystallization and preliminary X-ray analysis of the XMT and DXMT N-methyltransferases from Coffea canephora (robusta).
Caffeine is a secondary metabolite produced by a variety of plants including Coffea canephora (robusta) and there is growing evidence that caffeine is part of a chemical defence strategy protecting young leaves and seeds from potential predators. The genes encoding XMT and DXMT, the enzymes from Coffea canephora (robusta) that catalyse the three independent N-methyl transfer reactions in the caffeine-biosynthesis pathway, have been cloned and the proteins have been expressed in Escherichia coli. Both proteins have been crystallized in the presence of the demethylated cofactor S-adenosyl-L-cysteine (SAH) and substrate (xanthosine for XMT and theobromine for DXMT). The crystals are orthorhombic, with space group P2(1)2(1)2(1) for XMT and C222(1) for DXMT. X-ray diffraction to 2.8 A for XMT and to 2.5 A for DXMT have been collected on beamline ID23-1 at the ESRF.
McCarthy
Andrew A
AA
European Molecular Biology Laboratory, 6 Rue Jules Horowitz, BP 181, 38042 Grenoble, France. andrewmc@embl.fr
Biget
Laurent
L
Lin
Chenwei
C
Petiard
Vincent
V
Tanksley
Steve D
SD
McCarthy
James G
JG
D001483:Base Sequence; D003001:Cloning, Molecular; D040503:Coffea / Q000201:enzymology*; D003460:Crystallization; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D008780:Methyltransferases