Reaction mechanism of recombinant 3-oxoacyl-(acyl-carrier-protein) synthase III from Cuphea wrightii embryo, a fatty acid synthase type II condensing enzyme.
A unique feature of fatty acid synthase (FAS) type II of higher plants and bacteria is 3-oxoacyl-[acyl-carrier-protein (ACP)] synthase III (KAS III), which catalyses the committing condensing reaction. Working with KAS IIIs from Cuphea seeds we obtained kinetic evidence that KAS III catalysis follows a Ping-Pong mechanism and that these enzymes have substrate-binding sites for acetyl-CoA and malonyl-ACP. It was the aim of the present study to identify these binding sites and to elucidate the catalytic mechanism of recombinant Cuphea wrightii KAS III, which we expressed in Escherichia coli. We engineered mutants, which allowed us to dissect the condensing reaction into three stages, i.e. formation of acyl-enzyme, decarboxylation of malonyl-ACP, and final Claisen condensation. Incubation of recombinant enzyme with [1-(14)C]acetyl-CoA-labelled Cys(111), and the replacement of this residue by Ala and Ser resulted in loss of overall condensing activity. The Cys(111)Ser mutant, however, still was able to bind acetyl-CoA and to catalyse subsequent binding and decarboxylation of malonyl-ACP to acetyl-ACP. We replaced His(261) with Ala and Arg and found that the former lost activity, whereas the latter retained overall condensing activity, which indicated a general-base action of His(261). Double mutants Cys(111)Ser/His(261)Ala and Cys(111)Ser/His(261)Arg were not able to catalyse overall condensation, but the double mutant containing Arg induced decarboxylation of [2-(14)C]malonyl-ACP, a reaction indicating the role of His(261) in general-acid catalysis. Finally, alanine scanning revealed the involvement of Arg(150) and Arg(306) in KAS III catalysis. The results offer for the first time a detailed mechanism for a condensing reaction catalysed by a FAS type II condensing enzyme.
Abbadi
A
A
Institut für Biochemie, Universität Münster, Wilhelm-Klemm-Strasse 2, D-48149 Münster, Germany.
Brummel
M
M
Schütt
B S
BS
Slabaugh
M B
MB
Schuch
R
R
Spener
F
F
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D064429:Fatty Acid Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D019684:Magnoliopsida / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; D064429:Fatty Acid Synthases