Long-chain-fatty-acid—CoA ligase

The long chain fatty acyl-CoA ligase (or synthetase) is an enzyme (EC 6.2.1.3) of the ligase family that activates the oxidation of complex fatty acids. Long chain fatty acyl-CoA synthetase catalyzes the formation of fatty acyl-CoA by a two-step process proceeding through an adenylated intermediate. The enzyme catalyzes the following reaction,

Fatty acid + CoA + ATP ⇌ Acyl-CoA + AMP + PPi It is present in all organisms from bacteria to humans. It catalyzes the pre-step reaction for β-oxidation of fatty acids or can be incorporated in phospholipids.

Function Long chain fatty acyl-CoA synthetase, LC-FACS, plays a role in the physiological regulation of various cellular functions via the production of long chain fatty acyl-CoA esters, which reportedly have affected protein transport, enzyme activation, protein acylation, cell signaling, and transcriptional regulation. The formation of fatty acyl-CoA is catalyzed in two steps: a stable intermediate of fatty acyl-AMP molecule and then the product is formed—fatty acid acyl-CoA molecule. Fatty acyl CoA synthetase catalyzes the activation of a long fatty acid chain to a fatty acyl CoA, requiring the energy of 1 ATP to AMP and pyrophosphate. This step uses 2 "ATP equivalents" because pyrophosphate is cleaved into 2 molecules of inorganic phosphate, breaking a high-energy phosphate bond.

Mechanism and active site The mechanism for Long Chain Fatty Acyl-CoA Synthetase is a “bi uni uni bi ping-pong” mechanism. The uni and bi prefixes refer to the number of substrates that enter the enzyme and the number of products that leave the enzyme; bi describes a situation where two substrates enter the enzyme at the same time. Ping-pong signifies that a product is released before another substrate can bind to the enzyme. In step one, ATP and a long chain fatty acid enter the enzyme's active site. Within the active site the negatively charged oxygen on the fatty acid attacks the alpha phosphate on ATP, forming an ATP-long chain fatty acid intermediate. (Step 1, Figure 2) In the second step, Pyrophosphate (PPi) leaves, resulting in an AMP-long chain fatty acid molecule within the enzyme's active site. (Step 2, Figure 2) Coenzyme A now enters the enzyme and another intermediate is formed which consists of AMP-long chain fatty acid-Coenzyme A. (Step 3, Figure 2) At the end of this mechanism two products are released, AMP and acyl coa product. (Step 4, Figure 2) Acyl CoA is formed from long chain fatty acids through an acyl substitution. In an ATP dependent reaction, the fatty acid carboxylate is converted to a thioester. The final products of this reaction are acyl-CoA, pyrophosphate (PPi) and AMP.

Structure There are several highly conserved areas and a 20-30% amino acid sequence similarity between the members of this superfamily. The enzymes in the family consist of a large N-terminal and a small C-terminal domain, with the catalytic site positioned between the two domains. Substrate binding may affect the relative positions of the C- and N-terminal domains. The C-terminal domain of LC-FACS is assumed to be in an open conformation when a substrate is absent and in a closed conformation when a substrate is bound. The accessibility of the active site to solvent is reduced when the C- and N-terminal domains approach one another. The structure-function relationship between LC-FACS and the formation and processing of the acyl-AMP intermediate was still unclear. A domain swapped dimer is formed by LC-FACS, with monomer interacting at the N-terminal domains. A large electrostatically positive concave is located at the back of the structure in the central valley of the homodimer. Asp15 forms an intermolecular salt bridge with Arg176 in the dimer interactions. An intermolecular hydrogen bond is formed between the main chain carbonyl group of Glu16and the side chain of Arg199. At the interface, Glu175 forms an intermolecular salt bridge with Arg199. The L motif, a six-amino acid peptide linker, connects the large N-terminal domain and a small C-terminal domain of each LC-FACS monomer. The N-terminal domain is composed of two subdomains: a distorted antiparallel β-barrel and two β-sheets surrounded by α-helices forming an αβαβα sandwich. The small C-terminal globular domain consists of two-stranded β-sheet and a three-stranded antiparallel β-sheet flanked by three α-helices.

Dimer interaction The dimerization of LC-FACS is stabilized through a salt bridge between Asp15 of sequence A and Arg176 of sequence B. Figure 3 shows this salt bridge between these two amino acids. The yellow line between Asp15 and Arg176 shows the salt bridge present.

ATP binding to the C-terminal domain The conformations of the C-terminal domain of the LC-FACS structures are dependent on the presence of a ligand. AMP-PNP, a nonhydrolyzable ATP analogue, bound to LC-FACS results in the closed conformation with the C- and N-terminal domains directly interacting. In crystal structures, AMP-PNP is bound in a crevasse of each monomer at the interface between the N- and C-terminal domains. The closed conformation of the C-terminal domain is retained with myristroyl-AMP. Three residues in the C-terminal domain, Glu443, Glu475, and Lys527, interact noncovalently with L motif residues and the N-terminal domain to stabilize the closed conformation. There are two types of open conformations in the C-terminal domains of the uncomplexed structure. The C- and N-terminal domains do not interact directly for both monomers of the dimer. An extensive hydrogen bond network is used by the AMP moiety of the bound ATP molecule to hold the C- and N-terminal domains together.

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