Molecular cloning and expression of a novel cholinephosphotransferase involved in glycoglycerophospholipid biosynthesis of Mycoplasma fermentans.
A gene, mf1, encoding a novel cholinephosphotransferase in glycoglycerophospholipid (GGPL) biosynthesis of Mycoplasma fermentans PG18 was identified by genomic analysis, cloned, and expressed in Escherichia coli. The mf1 gene comprises an open reading frame of 777 bp encoding 258 amino acids. The mf1 gene product, Mf1, has 23% amino acid homology with LicD of Haemophilus influenzae but no homology with genes of other Mycoplasma species in the GenBank database. The reaction product of Mf1 using alpha-glucopyranosyl-1,2-dipalmitoilglycerol and cytidine 5'-diphosphocholine (CDP-choline) as substrates showed the specific protonated molecule at m/z 896, which corresponded to GGPL-I as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, the product ions of choline, phosphocholine, and hexose-bound phosphocholine were detected by tandem mass spectrometry (MS) analysis of protonated molecules at m/z 896. These results identified mf1 as a novel cholinephosphotransferase and showed that the phosphocholine transfer step is involved in the GGPL biosynthesis pathway of M. fermentans. This is the first report of a GGPL biosynthesis enzyme.
Ishida
Noriko
N
Laboratory of Applied Microbiology, Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Sapporo 060-8589, Japan. ishidan@chem.agr.hokudai.ac.jp
Irikura
Daisuke
D
Matsuda
Kazuhiro
K
Sato
Seiji
S
Sone
Teruo
T
Tanaka
Michiko
M
Asano
Kozo
K
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D002798:Diacylglycerol Cholinephosphotransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005994:Glycerophosphates / Q000096:biosynthesis* / Q000737:chemistry; D008969:Molecular Sequence Data; D016858:Mycoplasma fermentans / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D010802:Phylogeny; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005994:Glycerophosphates; D002798:Diacylglycerol Cholinephosphotransferase