Cloning, expression, purification, crystallization and preliminary X-ray analysis of NodS N-methyltransferase from Bradyrhizobium japonicum WM9.
The Nod factor (NF) is a rhizobial signal molecule that is involved in recognition of a legume host and the formation of root and stem nodules. Some unique enzymes are involved in the biosynthesis of NF, which is a variously but specifically substituted lipochitooligosaccharide. One of these enzymes is NodS, an N-methyltransferase that methylates end-deacetylated chitooligosaccharide substrates. In the methylation reaction, NodS uses S-adenosyl-L-methionine (SAM) as a methyl donor. To date, no structural information is available about NodS from any rhizobium. X-ray crystallographic studies of the NodS protein from Bradyrhizobium japonicum WM9, which infects the legumes lupin and serradella, have been undertaken. The nodS gene was cloned and the recombinant protein was expressed in Escherichia coli cells using natural amino acids and as an SeMet derivative. NodS without ligands was crystallized in the presence of PEG 3350 and MgCl(2). The protein was also crystallized in complex with S-adenosyl-L-homocysteine (SAH) in the presence of PEG 8000 and MgCl(2). SAH is produced from SAM as a byproduct of the methylation reaction. The crystals of apo NodS are tetragonal and diffracted X-rays to 2.42 A resolution. The NodS-SAH complex crystallizes in an orthorhombic space group and the crystals diffracted X-rays to 1.85 A resolution.
Cakici
Ozgur
O
Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
Sikorski
Michal
M
Stepkowski
Tomasz
T
Bujacz
Grzegorz
G
Jaskolski
Mariusz
M
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D020369:Bradyrhizobium / Q000201:enzymology*; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D008780:Methyltransferases