Biosynthesis of branched-chain fatty acid in bacilli: FabD (malonyl-CoA:ACP transacylase) is not essential for in vitro biosynthesis of branched-chain fatty acids.
It was found that the partially purified beta-ketoacyl-ACP synthase of Bacillus insolitus did not require the addition of FabD (malonyl-CoA:ACP transacylase, MAT) for the activity assay. This study therefore examined the necessity of FabD protein for in vitro branched-chain fatty acid (BCFA) biosynthesis by crude fatty acid synthetases (FAS) of Bacilli. To discover the involvement of FabD in the BCFA biosynthesis, the protein was removed from the crude FAS by immunoprecipitation. The His-tag fusion protein FabD of Bacillus subtilis was expressed in Escherichia coli and used for the preparation of antibody. The rabbit antibody raised against the expressed fusion protein specifically recognized the FabD in the crude FAS of B. subtilis. Evaluation of the efficacy of the immunoprecipitation showed that a trace of FabD protein was present in the antibody-treated crude FAS. However, this complete removal of FabD from the crude FAS did not abolish its BCFA biosynthesis, but only reduced the level to 50-60% of the control level for acyl-CoA primer and to 80% for alpha-keto-beta-methylvalerate primer. Furthermore, the FabD concentration did not necessarily correlate with the MAT specific activity in the enzyme fractions, suggesting the presence of another enzyme source of MAT activity. This study, therefore, suggests that FabD is not the sole enzyme source of MAT for in vitro BCFA biosynthesis, and implies the existence of a functional connection between fatty acid biosynthesis and another metabolic pathway.
Division of Molecular Biotechnology, Center of Molecular Bioscience, University of the Ryukyus, Nishihara, Okinawa, Japan. okuhiros@comb.u-ryukyu.ac.jp
Hirosuke
H
Oku
Naoya
N
Futamori
Kenichi
K
Masuda
Yumiko
Y
Shimabukuro
Tomoyo
T
Omine
Hironori
H
Iwasaki
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000378:metabolism; D051080:Acyl-Carrier Protein S-Malonyltransferase; D000217:Acyltransferases / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology*; D000906:Antibodies; D001407:Bacillus / Q000201:enzymology / Q000378:metabolism*; D001426:Bacterial Proteins; D064429:Fatty Acid Synthases / Q000378:metabolism; D005227:Fatty Acids / Q000096:biosynthesis*; D007118:Immunoassay; D011233:Precipitin Tests
D000906:Antibodies; D001426:Bacterial Proteins; D005227:Fatty Acids; D000217:Acyltransferases; D051080:Acyl-Carrier Protein S-Malonyltransferase; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; D064429:Fatty Acid Synthases