Purification, crystallization and preliminary crystallographic analysis of very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegans

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010 Apr 1;66(Pt 4):426-30. doi: 10.1107/S1744309110005002. Epub 2010 Mar 31.

Abstract

Acyl-CoA dehydrogenase [acyl-CoA:(acceptor) 2,3-oxidoreductase; EC 1.3.99.3] catalyzes the first reaction step in mitochondrial fatty-acid beta-oxidation. Here, the very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegans (cVLCAD) has been cloned and overexpressed in Escherichia coli strain BL21 (DE3). Interestingly, unlike other very-long-chain acyl-CoA dehydrogenases, cVLCAD was found to form a tetramer by size-exclusion chromatography coupled with in-line static light-scattering, refractive-index and ultraviolet measurements. Purified cVLCAD (12 mg ml(-1)) was successfully crystallized by the hanging-drop vapour-diffusion method under conditions containing 100 mM Tris-HCl pH 8.0, 150 mM sodium chloride, 200 mM magnesium formate and 13% PEG 3350. The crystal has a tetragonal form and a complete diffraction data set was collected and processed to 1.8 A resolution. The crystal belonged to space group C2, with unit-cell parameters a = 138.6, b = 116.7, c = 115.3 A, alpha = gamma = 90.0, beta = 124.0 degrees . A self-rotation function indicated the existence of one noncrystallographic twofold axis. A preliminary molecular-replacement solution further confirmed the presence of two molecules in one asymmetric unit, which yields a Matthews coefficient V(M) of 2.76 A(3) Da(-1) and a solvent content of 55%.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyl-CoA Dehydrogenase, Long-Chain / chemistry*
  • Acyl-CoA Dehydrogenase, Long-Chain / isolation & purification
  • Animals
  • Caenorhabditis elegans / enzymology*
  • Crystallization
  • Crystallography, X-Ray
  • Molecular Weight

Substances

  • Acyl-CoA Dehydrogenase, Long-Chain