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The malonyl‐CoA:ACP transacylase R117A variant catalyzes acyl‐transfer with a broad range of acyl‐CoA substrates
Author(s) -
Marcella Aaron M,
Barb Adam W
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.781.20
Subject(s) - chemistry , enzyme , substrate (aquarium) , biochemistry , mutagenesis , catalysis , malonyl coa , stereochemistry , acyl carrier protein , biosynthesis , mutant , gene , beta oxidation , biology , ecology
Prokaryotic fatty acid biosynthesis is catalyzed by multiple dissociated enzymes and represents a target for pathway engineering to generate novel biorenewable chemicals. We probed the active site of the E. coli malonyl‐CoA:ACP transacylase (FabD) with site directed mutagenesis to increase the substrate recognition profile. One single substitution, R117A, was predicted to disrupt anionic interaction with the malonate enzyme intermediate as well as several hydrogen bonds and allow for the entry of nonpolar and branched CoA substrates. We developed a rapid novel discontinuous fluorescence assay and probed FabD plus FabD R117A activity by measuring specific activities. FabD R117A efficiently transfers acetyl‐ (120 nmoles min −1 mg −1 ), acetoacetyl‐(60), propionyl‐(52), butyryl‐(11), isobutyryl‐(4), succinyl‐(16), and methylmalonyl‐ (3200) thioesters, unlike the wild‐type FabD that prefers malonyl‐CoA (4 mmoles min −1 mg −1 ) over methylmalonyl‐CoA(0.27) and acetoacetyl‐CoA (2 nmoles min −1 mg −1 ). K M values for FabD R117A vary slightly when compared to the wild type enzyme with malonyl‐CoA indicating catalytic rates are decreased. In total, these data suggest the FabD R117A variant represents a starting point for catalyst design to synthesize novel biorenewable molecules. Support or Funding Information National Science Foundation under Award No. EEC‐0813570 Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology at Iowa State University