Structure-function studies of yeast C-4 sphingolipid long chain base hydroxylase.
The roles of putative active site residues of the Saccharomyces cerevisiae sphingolipid C-4 long chain base hydroxylase (Sur2p) were investigated by site-directed mutagenesis. The replacement of any one of conserved His residues of three histidine-rich motifs with an alanine eliminated hydroxylase activity in vivo and in vitro, indicating that they are all essential elements of the active site. An additional conserved His residue (His 249) outside of the histidine-rich cluster region was also found to be crucial for activity. Additional mutants altered in residues in close proximity to the histidine-rich cluster were generated. In order to determine their roles in hydroxylase vs. desaturase activities, residues were replaced with conserved residues from the yeast Delta7-sterol-C5(6)-desaturase, Erg3p. Residues Phe 174, Asn 182, Ser 191, Leu 196, Pro 199, Asn 266, Tyr 269, Asp 271 and Gln 275 appear to be additionally important elements of the active site but their conversion into corresponding Erg3p residues did not lead to a gain in desaturase activity. It is concluded that Sur2p is a membrane-bound hydroxylase that belongs to the diiron family of eight-histidine motif enzymes.
Idkowiak-Baldys
Jolanta
J
Department of Biology, Utah State University, Logan, UT 84322-5305, USA.
Takemoto
Jon Y
JY
Grilley
Michelle M
MM
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology; D006639:Histidine / Q000378:metabolism; D006899:Mixed Function Oxygenases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010456:Peptides, Cyclic / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D013107:Sphingolipids / Q000378:metabolism*; D013329:Structure-Activity Relationship
D000935:Antifungal Agents; D010456:Peptides, Cyclic; D029701:Saccharomyces cerevisiae Proteins; D013107:Sphingolipids; C105246:syringomycin E; D006639:Histidine; D006899:Mixed Function Oxygenases; C109313:SUR2 protein, S cerevisiae