Abstract
The synthases that produce fatty acids in mammals (FASs) are arranged as large multidomain polypeptides. The growing fatty acid chain is bound covalently during chain elongation and reduction to the acyl carrier protein (ACP) domain that is then able to access each catalytic site. In this work we report the high-resolution nuclear magnetic resonance (NMR) solution structure of the isolated rat fatty acid synthase apoACP domain. The final ensemble of NMR structures and backbone (15)N relaxation studies show that apoACP adopts a single, well defined fold. On conversion to the holo form, several small chemical shift changes are observed on the ACP for residues surrounding the phosphopantetheine attachment site (as monitored by backbone (1)H-(15)N correlation experiments). However, there are negligible chemical shift changes when the holo form is modified to either the hexanoyl or palmitoyl forms. For further NMR analysis, a (13)C,(15)N-labeled hexanoyl-ACP sample was prepared and full chemical shift assignments completed. Analysis of two-dimensional F(2)-filtered and three-dimensional (13)C-edited nuclear Overhauser effect spectroscopy experiments revealed no detectable NOEs to the acyl chain. These experiments demonstrate that unlike other FAS ACPs studied, this Type I ACP does not sequester a covalently linked acyl moiety, although transient interactions cannot be ruled out. This is an important mechanistic difference between the ACPs from Type I and Type II FASs and may be significant for the modulation and regulation of these important mega-synthases.
Highlights
Fatty acids in mammals, utilize simple acyl units, bound to the phosphopantetheine arm of a holo acyl carrier protein (ACP) domain, for chain initiation and elongation
Spinach fatty acids in mammals (FASs) holo-ACP exists in equilibrium between a folded and largely disordered form, upon acylation this equilibrium is shifted toward the folded form
We have previously reported the low resolution solution phase structure of the isolated rat FAS apoACP domain [10] and found it to adopt an ␣ helical bundle structure consistent with the Type II ACPs from fatty acid [11] and polyketide synthases (PKSs) [12]
Summary
Protein Expression and Purification—Rat FAS apoACP was recombinantly expressed from E. coli strain BL21(DE3) carrying the rat FAS ACP (amino acids 2114 –2202) in the pET15b expression plasmid as previously described [13]. NMR of Rat FAS Hexanoyl-ACP Domain—A 13C,15N dual acquisition (CN) NOESY experiment (acquired with 150 ms mixing time) and a two-dimensional F2-filtered NOESY experiment with a 400-ms mixing time [19] were recorded on a 1.5 mM sample of [12C,14N]-hexanoyl-[13C,15N]-labeled rat FAS apoACP. In this sample both the hexanoyl group and phosphopantetheine side arm are not enriched with 13C or 15N. Uncertainties in the NOE values were estimated from the base plane noise in two-dimensional 1H-15N HSQC spectra recorded with and without proton saturation according to Farrow et al [26]. Itive ion mode on a QStar XL mass spectrometer (Applied Biosystems)
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