No abstract

Acetyl-coenzyme A carboxylases (ACCs) have crucial roles in fatty acid metabolism. Soraphen A, a macrocyclic polyketide natural product, is a nanomolar inhibitor against the biotin carboxylase (BC) domain of human, yeast, and other eukaryotic ACCs. Here we report the crystal structures of the yeast BC domain, alone and in complex with soraphen A. Soraphen has extensive interactions with an allosteric site, about 25 A from the active site. The specificity of soraphen is explained by large structural differences between the eukaryotic and prokaryotic BC in its binding site, confirmed by our studies on the effects of single-site mutations in this binding site. Unexpectedly, our structures suggest that soraphen may bind in the BC dimer interface and inhibit the BC activity by disrupting the oligomerization of this domain. Observations from native gel electrophoresis confirm this structural insight. The structural information provides a foundation for structure-based design of new inhibitors against these enzymes.

Ionic liquids are described that contain duplex DNA as the anion and polyether-decorated transition metal complexes based on M(MePEG-bpy)(3)(2+) as the cation (M = Fe, Co; MePEG-bpy = 4,4'-(CH(3)(OCH(2)CH(2))(7)OCO)(2)-2,2'-bipyridine). When the undiluted liquid DNA-or molten salt-is interrogated electrochemically by a microelectrode, the molten salts exhibit cyclic voltammograms due to the physical diffusion (D(PHYS)) of the polyether-transition metal complex. When M = Co(II), the cyclic voltammogram of the melt shows an oxidative wave due to the Co(III/II) couple at E(1/2) = 0.40 V (versus Ag/AgCl) and a D(PHYS) of 6 x 10(-12) cm(2)/s, which is significantly lower than that for Co(MePEG-bpy)(3)(ClO(4))(2) (D(PHYS) = 2.6 x 10(-10) cm(2)/s) due to greater viscosity provoked by the DNA polymer. When a 1:1 mixture is made of the Co(MePEG-bpy)(3).DNA and Fe(MePEG-bpy)(3)(ClO(4))(2) melts, two redox waves are observed. The first is due to the Co(III/II) couple, and the second is a catalytic wave due to oxidation of guanine in DNA by electrogenerated Fe(III) in the undiluted melt. Independent experiments show that the Fe(III) form of the complex selectively oxidizes guanine in duplex DNA. These DNA molten salts constitute a new class of materials whose properties can be controlled by nucleic acid sequence and that can be interrogated in undiluted form on microelectrode arrays.

The proton-coupled electron-transfer reactions of guanine and other modified purine nucleobases have been investigated by electrochemistry and stopped-flow spectrophotometry. The rate of oxidation of guanine by Ru(bpy) 3 3+ varied linearly with the fraction of D 2 O in H 2 O/D 2 O mixtures (bpy ) 2,2′-bipyridine), suggesting the involvement of a single proton, consistent with earlier studies (Weatherly, S. C.; Yang, I. V.; Thorp, H. H. J. Am. Chem. Soc. 2001, 123, 1236-1237. The oxidation of the tetrabutylammonium salt of 2′-deoxyguanosine-5′-monophosphate was investigated in acetonitrile. The oxidants were polypyridyl complexes of Ru(III) and Fe(III) that ranged in oxidation potential from 0.83 to 1.6 V versus SCE. The values of (RT ln k) obtained for these reactions increased with driving force with a slope of 0.5 ( 0.1, consistent with a simple electron-transfer reaction. A conventional slope of 0.5 was not observed in aqueous solution where deprotonation of the guanine nucleobase was feasible. In addition to the driving force dependence, there was a dramatic suppression in the overall rate of guanine oxidation in acetonitrile compared to water both for dilute solution and for modified electrodes. The driving force dependence for the rate of oxidation of 7-deazaguanine and 7-deazaadenine was also investigated; plots of (RT ln k) versus E 1/2 showed slopes of 1.1 in aqueous solution, and these reactions gave large isotope effects ranging from 2.2 to 10, depending on the nucleobase and the oxidant. In contrast, the base 7,8-dihydro-8-oxoguanine did not exhibit an isotope effect, because the pK a of this nucleobase is known to be near 7. Taken together, these results suggest a picture where oxidation of guanine in aqueous solution by these oxidants proceeds via concerted proton-coupled electron transfer.

Acetyl-CoA carboxylase (ACC) catalyses the first step in fatty-acid biosynthesis. Owing to its role in primary metabolism, ACC has been exploited as a commercial herbicide target and identified as a chemically validated fungicide target. In animals, ACC is also a key regulator of fat metabolism. This function has made ACC a prime target for the development of anti-obesity and anti-Type II diabetes therapeutics. Despite its economic importance, there is a lack of published information on recombinant expression of ACC. We report here the expression of enzymically active fungal (Ustilago maydis ) ACC in Escherichia coli. The recombinant enzyme exhibited Km values of 0.14+/-0.013 mM and 0.19+/-0.041 mM for acetyl-CoA and ATP respectively, which are comparable with those reported for the endogenous enzyme. The polyketide natural product soraphen is a potent inhibitor of the BC (biotin carboxylase) domain of endogenous fungal ACC. Similarly, recombinant ACC activity was inhibited by soraphen with a K(i) of 2.1+/-0.9 nM. A truncated BC domain that included amino acids 2-560 of the full-length protein was also expressed in E. coli. The isolated BC domain was expressed to higher levels, and was more stable than full-length ACC. Although incapable of enzymic turnover, the BC domain exhibited high-affinity soraphen binding (Kd 1.1+/-0.3 nM), demonstrating a native conformation. Additional BC domains from the phytopathogenic fungi Magnaporthe grisea and Phytophthora infestans were also cloned and expressed, and were shown to exhibit high-affinity soraphen binding. Together, these reagents will be useful for structural studies and assay development.