ABSTRACT
Crystal structure data for Escherichia coli beta-ketoacyl synthase (KAS) I with C(10) and C(12) fatty acid substrates bound in conjunction with results from mutagenizing residues in the active site leads to a model for catalysis. Differences from and similarities to the other Claisen enzymes carrying out decarboxylations reveal two catalytic mechanisms, one for KAS I and KAS II, the other for KAS III and chalcone synthase. A comparison of the structures of KAS I and KAS II does not reveal the basis of chain-length specificity. The structures of the Arabidopsis thaliana KAS family are compared.
Subject(s)
3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/chemistry , 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/metabolism , Isoenzymes/chemistry , Isoenzymes/metabolism , Arabidopsis/enzymology , Binding Sites , Catalysis , Dimerization , Escherichia coli/enzymology , Models, Molecular , Peroxisomes/enzymology , Protein Conformation , Protein Structure, Secondary , Saccharomyces cerevisiae/enzymology , Substrate SpecificityABSTRACT
Escherichia coli beta-ketoacyl synthases (KAS) I and II carry out the elongation steps in fatty acid synthesis. Analyses using the cross-linker BS(3) [bis(sulphosuccinimidyl) suberate] and surface-enhanced laser desorption/ionization-time-of-flight MS disclosed only monomeric and dimeric forms of KAS II, whereas KAS I also forms higher multimers. The binding affinities for KAS I and KAS II to C(14)-acyl carrier protein (ACP) as well as for C(14)-ACP to KAS I and KAS II were determined. KAS I is sensitive to the ACP released during the transfer reaction, with 50% inhibition at 0.17 microM ACP close to the physiological concentration of ACP (0.13 microM). KAS I and II also differ in carrying out the decarboxylation step of the elongation reaction.
Subject(s)
3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/metabolism , Acyl Carrier Protein/metabolism , Isoenzymes/metabolism , 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/antagonists & inhibitors , 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/chemistry , Acyl Carrier Protein/antagonists & inhibitors , Acyl Carrier Protein/chemistry , Binding Sites , Cross-Linking Reagents/pharmacology , Escherichia coli/enzymology , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Kinetics , Myristic Acid/metabolism , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Succinimides/pharmacologyABSTRACT
Site-directed mutagenesis and detailed fluorescence studies were used to study the structure and dynamics of recombinant human proapolipoprotein (proapo) A-I in the lipid free state and in reconstituted high-density lipoprotein (rHDL) particles. Five different mutants of proapoA-I, each containing a single tryptophan residue, were produced in bacteria corresponding to each of the naturally occurring Trp residues (position -3 in the pro-segment, 8, 50, 72, and 108) in the N-terminal half of the protein. Structural analyses indicated that the conservative Phe-Trp substitutions did not perturb the conformation of the mutants with respect to the wild-type protein. Steady-state fluorescence studies indicated that all of the Trp residues exist in nonpolar environments that are highly protected from solvent in both the lipid-free and lipid-bound forms. Time-resolved lifetime and anisotropy studies indicated that the shape of the monomeric form of proapoA-I is a prolate ellipsoid with an axial ratio of about 6:1. In addition, the region surrounding Trp 108 appears to be more mobile than the rest of the protein in the lipid-free state. However, in rHDL particles, no significant domain motion was detected for any of the Trp residues. The results presented in this work are consistent with a model for monomeric lipid-free proapoA-I in which the N-terminal half of the molecule is organized into a bundle of helices.
