Primary structure, regioselectivity, and evolution of the membrane-bound fatty acid desaturases of Claviceps purpurea.
Two cDNAs with sequence similarity to fatty acid desaturase genes were isolated from the phytopathogenic fungus, Claviceps purpurea. The predicted amino acid sequences of the corresponding genes, named CpDes12 and CpDesX, share 87% identity. Phylogenetic analysis indicates that CpDes12 and CpDesX arose by gene duplication of an ancestral Delta(12)-desaturase gene after the divergence of Nectriaceae and Clavicipitaceae. Functional expression of CpDes12 and CpDesX in yeast (Saccharomyces cerevisiae) indicated that CpDes12 is primarily a "Delta(12)"-desaturase, whereas CpDesX is a novel desaturase catalyzing "Delta(12)," "Delta(15)," and "omega(3)" types of desaturation with omega(3) activity predominating. CpDesX sequentially desaturates both 16:1-9c and 18:1-9c to give 16:3-9c,12c,15c and 18:3-9c,12c,15c, respectively. In addition, it could also act as an omega(3)-desaturase converting omega(6)-polyunsaturates 18:3-6c,9c,12c, 20:3-8c,11c,14c, and 20:4-5c,8c,11c,14c to their omega(3) counterparts 18:4-6c,9c,12c,15c, 20:4-8c,11c,14c,17c, and 20:5-5c,8c,11c,14c,17c, respectively. By using reciprocal site-directed mutagenesis, we demonstrated that two residues (isoleucine at 152 and alanine at 206) are critical in defining the catalytic specificity of these enzymes and the C-terminal amino acid sequence (residues 302-477) was also found to be important. These data provide insights into the nature of regioselectivity in membrane-bound fatty acid desaturases and the relevant structural determinants. The authors suggest that the regios-electivity of such enzymes may be best understood by considering the relative importance of more than one regioselective preference. In this view, CpDesX is designated as anu + 3(omega(3)) desaturase, which primarily references an existing double bond (nu + 3 regioselectivity) and secondarily shows preference for omega(3) desaturation.
Meesapyodsuk
Dauenpen
D
Department of Applied Microbiology and Food Science, University of Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan, Canada.
Reed
Darwin W
DW
Covello
Patrick S
PS
Qiu
Xiao
X
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D002967:Claviceps / Q000201:enzymology* / Q000235:genetics; D019143:Evolution, Molecular; D044943:Fatty Acid Desaturases / Q000235:genetics / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity / Q000235:genetics
D005231:Fatty Acids, Unsaturated; D005656:Fungal Proteins; D011994:Recombinant Proteins; D044943:Fatty Acid Desaturases