A KAS2 cDNA complements the phenotypes of the Arabidopsis fab1 mutant that differs in a single residue bordering the substrate binding pocket.
The fab1 mutant of Arabidopsis is partially deficient in activity of beta-ketoacyl-[acyl carrier protein] synthase II (KAS II). This defect results in increased levels of 16:0 fatty acid and is associated with damage and death of the mutants at low temperature. Transformation of fab1 plants with a cDNA from Brassica napus encoding a KAS II enzyme resulted in complementation of both mutant phenotypes. The dual complementation by expression of the single gene proves that low-temperature damage is a consequence of altered membrane unsaturation. The fab1 mutation is a single nucleotide change in Arabidopsis KAS2 that results in a Leu337Phe substitution. The Leu337 residue is conserved among plant and bacterial KAS proteins, and in the crystal structures of E. coli KAS I and KAS II, this leucine abuts a phenylalanine whose imidazole ring extends into the substrate binding cavity causing the fatty acid chain to bend. For functional analysis the equivalent Leu207Phe mutation was introduced into the fabB gene encoding the E. coli KAS I enzyme. Compared to wild-type, the Leu207Phe protein showed a 10-fold decrease in binding affinity for the fatty acid substrate, exhibited a modified behavior during size-exclusion chromatography and was severely impaired in condensation activity. These results suggest that the molecular defect in fab1 plants is a structural instability of the KAS2 gene product induced by insufficient space for the imidazole ring of the mutant phenylalanine residue.
Carlsson
Anders S
AS
Institute of Biological Chemistry, Washington State University, Pullman 99164, USA.
LaBrie
Samuel T
ST
Kinney
Anthony J
AJ
von Wettstein-Knowles
Penny
P
Browse
John
J
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D029688:Brassica napus / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005227:Fatty Acids / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010641:Phenotype; D030821:Plants, Genetically Modified; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; D005227:Fatty Acids; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; C469227:KAS2 protein, Arabidopsis