Functional analysis of Chinese hamster phosphatidylserine synthase 1 through systematic alanine mutagenesis.
PtdSer (phosphatidylserine) synthesis in mammalian cells occurs through the exchange of L-serine with the base moieties of phosphatidylcholine and phosphatidylethanolamine, which is catalysed by PSS (PtdSer synthase) 1 and 2 respectively. PtdSer synthesis in intact cells and an isolated membrane fraction was inhibited by exogenous PtdSer, indicating that feedback control is involved in the regulation of PtdSer biosynthesis. PSS 1 and 2 are similar in amino acid sequence, with an identity of 32%; however, due to a lack of homology with other known enzymes, their amino acid sequences do not provide information on their catalytic and regulatory mechanisms. In the present study, to identify amino acid residues crucial for the activity and/or regulation of PSS 1, we systematically introduced mutations into a Chinese hamster PSS 1 cDNA clone; namely, each of the 66 polar amino acid residues common to PSS 2 was replaced with an alanine residue. On analysis of Chinese hamster ovary cells transfected with each of the alanine mutant clones, we identified eight amino acid residues (His-172, Glu-197, Glu-200, Asn-209, Glu-212, Asp-216, Asp-221 and Asn-226) as those crucial for the enzyme reaction or the maintenance of the correct structure required for serine base-exchange activity. Among these residues, Asn-209 was suggested to be involved in the recognition and/or binding of free L-serine. We also identified six amino acid residues (Arg-95, His-97, Cys-189, Arg-262, Gln-266 and Arg-336) as those important for regulation of PSS 1. In addition, we found that the alanine mutations at Tyr-111, Asp-166, Arg-184, Arg-323, and Glu-364 affected the production and/or stability of PSS 1 in Chinese hamster ovary cells.
Ohsawa
Tomoko
T
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan.
Nishijima
Masahiro
M
Kuge
Osamu
O
D000409:Alanine / Q000235:genetics / Q000502:physiology*; D000596:Amino Acids / Q000502:physiology; D000818:Animals; D010717:CDPdiacylglycerol-Serine O-Phosphatidyltransferase / Q000502:physiology*; D016466:CHO Cells / Q000201:enzymology; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics / Q000502:physiology*; D012694:Serine / Q000378:metabolism
D000596:Amino Acids; D012694:Serine; D010717:CDPdiacylglycerol-Serine O-Phosphatidyltransferase; D000409:Alanine