The authors have isolated and characterized a novel serine palmitoyltransferase (SPT)-like gene in marine Emiliania huxleyi virus (EhV-99B1). The open-reading frame (ORF) of EhV99B1-SPT encoded a protein of 496 amino acids with a calculated molecular mass of 96 kDa and Ip 6.01. The results of sequence analysis showed that there was about 31%-45% identity in amino acid sequence with other organisms. The maximum likelihood phylogenetic tree suggested that the EhV99B1-SPT gene possibly horizontally transferred from the eukaryote. Hydrophobic profiles of deduced amino acid sequences suggested a hydrophobic, globular and membrane-associated protein with five transmembrane domains (TMDs) motifs. Several potential N-linked glycosylation sites were presented in SPT. These results suggested that EhV99B1-SPT was an integral endoplasmic reticulum membrane protein. Despite lower sequence identity, the secondary and three-dimensional structures predicted showed that the "pocket" structure element composed of 2α-helices and 4β-sheets was the catalytic center of this enzyme, with a typical conserved "TFTKSFG" active site in the N-terminal region and was very close to those of prokaryotic organisms. However, the N-terminal domain of EhV99B1-SPT most closely resembled the LCB2 catalysis subunit and the C-terminal domain most closely resembled the LCB1 regulatory subunit of other organisms which together formed a spherical molecule. This "chimera" was highly similar to the prokaryotic homologous SPT. For a functional identification, the EhV99B1-LCB2 subunit gene was expressed in Escherichia coli, which resulted in significant accumulation of new sphingolipid in E. coli cells.
Keywords: Emiliania huxleyi virus; clone expressiong and characterization; serine palmitoyltransferase (SPT).
© The Chinese Society of Oceanography and Springer-Verlag Berlin Heidelberg 2012.