Enzymatic synthesis of Mycoplasma fermentans specific glycoglycerophospholipid from 1,2-dipalmitoylglycerol.
A gene, mf3, encoding glycosyltransferase in glycoglycerophospholipid (GGPL; GGPL-I and GGPL-III) biosynthesis in Mycoplasma fermentans PG18 was identified by genomic analysis, cloned, modified codon usage, and expressed in Escherichia coli. The mf3 gene consists of an open reading frame of 1221 bp encoding 406 amino acids. The mf3 gene product, Mf3, has 27% amino acid homology with glycosyltransferase of Borrelia burgdorferi but no homology to genes of other Mycoplasma species in the GenBank database. The reaction product of Mf3 using 1,2-dipalmitoilglycerol and UDP-glucose as substrates showed a specific sodium adducted ion at m/z 753, which corresponded to glucopyranosyl dipalmitoilglycerol as determined by MALDI-TOF mass spectrometry. Furthermore, in the reaction product by Mf3 and Mf1 which was a cholinephosphotransferase and previously cloned from M. fermentans PG18, an ion at m/z 896 corresponding to GGPL-I was detected by mass spectrometry. The product ions of choline, phosphocholine, and hexose-bound phosphocholine were detected by tandem MS analysis of protonated molecules at m/z 896. From these results, mf3 was identified as a glycosyltransferase. It was suggested that glucose transfer and phosphocholine transfer to 1,2-dipalmitoylglycerol are involved in the GGPL biosynthesis pathway of M. fermentans PG18.
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
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004075:Diglycerides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D006017:Glycolipids / Q000096:biosynthesis* / Q000737:chemistry; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D016858:Mycoplasma fermentans / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
C106372:1''-phosphocholine-2''-aminodihydroxypropane-3''-phospho-6'-glucopyranosyl-(1'-3)-1,2-dipalmitoylglycerol; D017931:DNA Primers; D004075:Diglycerides; D006017:Glycolipids; D011994:Recombinant Proteins; C091226:6'-O-phosphocholineglucopyranosyl-(1'-3)-1,2-dipalmitoyl; C005071:dipalmitin; D016695:Glycosyltransferases