Structural basis of the substrate-specific two-step catalysis of long chain fatty acyl-CoA synthetase dimer

J Biol Chem. 2004 Jul 23;279(30):31717-26. doi: 10.1074/jbc.M400100200. Epub 2004 May 15.

Abstract

Long chain fatty acyl-CoA synthetases are responsible for fatty acid degradation as well as physiological regulation of cellular functions via the production of long chain fatty acyl-CoA esters. We report the first crystal structures of long chain fatty acyl-CoA synthetase homodimer (LC-FACS) from Thermus thermophilus HB8 (ttLC-FACS), including complexes with the ATP analogue adenosine 5'-(beta,gamma-imido) triphosphate (AMP-PNP) and myristoyl-AMP. ttLC-FACS is a member of the adenylate forming enzyme superfamily that catalyzes the ATP-dependent acylation of fatty acid in a two-step reaction. The first reaction step was shown to propagate in AMP-PNP complex crystals soaked with myristate solution. Myristoyl-AMP was identified as the intermediate. The AMP-PNP and the myristoyl-AMP complex structures show an identical closed conformation of the small C-terminal domains, whereas the uncomplexed form shows a variety of open conformations. Upon ATP binding, the fatty acid-binding tunnel gated by an aromatic residue opens to the ATP-binding site. The gated fatty acid-binding tunnel appears only to allow one-way movement of the fatty acid during overall catalysis. The protein incorporates a hydrophobic branch from the fatty acid-binding tunnel that is responsible for substrate specificity. Based on these high resolution crystal structures, we propose a unidirectional Bi Uni Uni Bi Ping-Pong mechanism for the two-step acylation by ttLC-FACS.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Amino Acid Sequence
  • Base Sequence
  • Catalysis
  • Catalytic Domain
  • Coenzyme A Ligases / chemistry*
  • Coenzyme A Ligases / genetics
  • Coenzyme A Ligases / metabolism*
  • Crystallography, X-Ray
  • DNA Primers
  • DNA, Bacterial / genetics
  • Dimerization
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Thermus thermophilus / enzymology
  • Thermus thermophilus / genetics

Substances

  • DNA Primers
  • DNA, Bacterial
  • Recombinant Proteins
  • Adenosine Triphosphate
  • Coenzyme A Ligases

Associated data

  • PDB/1ULT
  • PDB/1V25
  • PDB/1V26