Active-site residues of a plant membrane-bound fatty acid elongase beta-ketoacyl-CoA synthase, FAE1 KCS.
The fatty acid elongase-1 beta-ketoacyl-CoA synthase, FAE1 KCS, a seed-specific elongase condensing enzyme from Arabidopsis, is involved in the production of eicosenoic (C20:1) and erucic (C22:1) acids. Alignment of the amino acid sequences of FAE1 KCS, KCS1, and five other putative elongase condensing enzymes (KCSs) revealed the presence of six conserved cysteine and four conserved histidine residues. Each of the conserved cysteine and histidine residues was individually converted by site-directed mutagenesis to both alanine and serine, and alanine and lysine respectively. After expression in yeast cells, the mutant enzymes were analyzed for their fatty acid elongase activity. Our results indicated that only cysteine 223 is an essential residue for enzyme activity, presumably for acyl chain transfer. All histidine substitutions resulted in complete loss of elongase activity. The loss of activity of these mutants was not due to their lower expression level since immunoblot analysis confirmed each was expressed to the same extent as the wild type FAE1 KCS.
Ghanevati
M
M
Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA.
Jaworski
J G
JG
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D003545:Cysteine / Q000737:chemistry; D004883:Erucic Acids / Q000378:metabolism; D000081031:Fatty Acid Elongases; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment
D004883:Erucic Acids; C049811:erucic acid; D006639:Histidine; D000123:Acetyltransferases; D000081031:Fatty Acid Elongases; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; D003545:Cysteine