Cloning and characterization of squalene synthase gene from Fusarium fujikuroi (Saw.) Wr.
The gene encoding squalene synthase (GfSQS) was cloned from Fusarium fujikuroi (Gibberella fujikuroi MP-C) and characterized. The cloned genomic DNA is 3,267 bp in length, including the 5'-untranslated region (UTR), 3'-UTR, four exons, and three introns. A noncanonical splice-site (CA-GG, or GC-AG) was found at the first intron. The open reading frame of the gene is 1,389 bp in length, corresponding to a predicted polypeptide of 462 amino acid residues with a MW 53.4 kDa. The predicted GfSQS shares at least four conserved regions involved in the enzymatic activity with the SQSs of varied species. The recombinant protein was expressed in E. coli and detected by SDS-PAGE and western blot. GC-MS analysis showed that the wild-type GfSQS could catalyze the reaction from farnesyl diphosphate (FPP) to squalene, while the mutant mGfSQS (D82G) lost total activity, supporting the prediction that the aspartate-rich motif (DTXED) in the region I of SQS is essential for binding of the diphosphate substrate.
Zhao
Rui-Yu
RY
Department of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Xian Nong Tan Street, Beijing, 100050, China.
Xiao
Wei
W
Cheng
Hai-Li
HL
Zhu
Ping
P
Cheng
Ke-Di
KD
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004271:DNA, Fungal / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005670:Fusarium / Q000201:enzymology / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013185:Squalene / Q000032:analysis
D004271:DNA, Fungal; D005656:Fungal Proteins; D011994:Recombinant Proteins; D013185:Squalene; D013186:Farnesyl-Diphosphate Farnesyltransferase