Characterization of cyclopropane fatty-acid synthase from Sterculia foetida.
Cyclopropane synthase from Sterculia foetida developing seeds catalyzes the addition of a methylene group from S-adenosylmethionine to the cis double bond of oleic acid (Bao, X., Katz, S., Pollard, M., and Ohlrogge, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7172-7177). To understand this enzyme better, differential expression in leaf and seed tissues, protein properties, and substrate preferences of plant cyclopropane synthase were investigated. Immunoblot analysis with antibodies raised to recombinant S. foetida cyclopropane synthase (SfCPA-FAS) revealed that SfCPA-FAS is expressed in S. foetida seeds, but not in leaves, and is a membrane protein localized to microsomal fractions. Transformed tobacco cells expressing SfCPA-FAS were labeled in vivo with L-[methyl-(14)C]methionine and assayed in vitro with S-adenosyl-L-[methyl-(14)C]methionine. These kinetic experiments demonstrated that dihydrosterculate was synthesized from oleic acid esterified at the sn-1 position of phosphatidylcholine (PC). Furthermore, analysis of acyl chains at sn-1 and sn-2 positions that accumulated in PC from S. foetida developing seeds and from tobacco cells expressing SfCPA-FAS also demonstrated that greater than 90% of dihydrosterculate was esterified to the sn-1 position. Thus, we conclude that SfCPA-FAS is a microsomal localized membrane protein that catalyzes the addition of methylene groups derived from S-adenosyl-L-methionine across the double bond of oleic acid esterified to the sn-1 position of PC. A survey of plant and bacterial genomes for sequences related to SfCPA-FAS indicated that a peptide domain with a putative flavin-binding site is either fused to the methyltransferase domain of the plant protein or is often found encoded by a gene adjacent to a bacterial cyclopropane synthase gene.
Bao
Xiaoming
X
Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824, USA.
Thelen
Jay J
JJ
Bonaventure
Gustavo
G
Ohlrogge
John B
JB
D002458:Cell Fractionation; D007700:Kinetics; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D030821:Plants, Genetically Modified / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D032361:Sterculia / Q000201:enzymology*; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology
D011994:Recombinant Proteins; D008780:Methyltransferases; C019964:cyclopropane synthetase