Enzymatic synthesis of Mycoplasma fermentans specific glycoglycerophospholipid from 1,2-dipalmitoylglycerol

• Published in: Journal of bioscience and bioengineering 2010-04, Vol.109 (4), p.341-345
• Main Authors: Ishida, Noriko, Irikura, Daisuke, Matsuda, Kazuhiro, Sato, Seiji, Sone, Teruo, Tanaka, Michiko, Asano, Kozo
• Format: Article
• Language: English
• Subjects: AMINO ACID SEQUENCES; Amino acids; Base Sequence; Bioconversions. Hemisynthesis; Biological and medical sciences; BIOSINTESIS; BIOSYNTHESE; BIOSYNTHESIS; Biotechnology; Borrelia burgdorferi; Diglycerides - metabolism; DNA Primers - genetics; ENZIMAS; ENZYME; ENZYMES; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; ESPECTROMETRIA DE MASAS; EXPRESION GENICA; EXPRESSION DES GENES; FOSFOLIPIDOS; Fundamental and applied biological sciences. Psychology; GENE EXPRESSION; Genes, Bacterial; GENETICALLY MODIFIED MICROORGANISMS; GLICOSILTRANSFERASAS; Glycerophospholipid; Glycolipids - biosynthesis; Glycolipids - chemistry; GLYCOSYLTRANSFERASE; GLYCOSYLTRANSFERASES; Glycosyltransferases - genetics; Glycosyltransferases - metabolism; http://www.fao.org/aos/agrovoc#c_11192; http://www.fao.org/aos/agrovoc#c_12421; http://www.fao.org/aos/agrovoc#c_2603; http://www.fao.org/aos/agrovoc#c_27527; http://www.fao.org/aos/agrovoc#c_34287; http://www.fao.org/aos/agrovoc#c_37202; http://www.fao.org/aos/agrovoc#c_5020; http://www.fao.org/aos/agrovoc#c_5803; http://www.fao.org/aos/agrovoc#c_928; MASS SPECTROMETRY; Methods. Procedures. Technologies; MICRO-ORGANISME MODIFIE GENETIQUE; microbial enzymes; MICROORGANISMOS MODIFICADOS GENETIC; MYCOPLASMA; Mycoplasma fermentans; Mycoplasma fermentans - enzymology; Mycoplasma fermentans - genetics; Mycoplasma fermentans - metabolism; PHOSPHATIDE; PHOSPHOLIPIDS; Phylogeny; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; SECUENCIAS DE AMINOACIDOS; SEQUENCE D'ACIDES AMINES; SPECTROMETRIE DE MASSE; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; Tandem Mass Spectrometry
• ISSN: 1389-1723; 1347-4421
• DOI: 10.1016/j.jbiosc.2009.09.049

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.