Identification of the Citrate-binding Site of Human ATP-Citrate Lyase Using X-ray Crystallography

Abstract

ATP-citrate lyase (ACLY, EC 2.3.3.8) 3 catalyzes the reaction, citrate ϩ CoA ϩ ATP 3 acetyl-CoA ϩ oxaloacetate ϩ ADP ϩ P i , in the presence of magnesium ions (1). ACLY is the cytoplasmic enzyme linking energy metabolism from carbohydrates to the production of fatty acids. Acetyl-CoA produced in the mitochondria cannot be exported to the cytoplasm. Instead, acetylCoA in mitochondria is transformed to citrate by citrate synthase, and citrate is exported to the cytoplasm where ACLY regenerates acetyl-CoA. This acetyl-CoA is an important precursor for fatty acid synthesis. In addition, ACLY has been shown to signal the metabolic state of cells, likely by providing acetyl-CoA for histone acetyltransferases in the cells' nuclei (2). The physiological importance of the enzyme is supported by knock-out experiments in which mice embryos lacking ATPcitrate lyase could not be obtained (3).Our understanding of the reaction mechanism of ACLY has been based on studies of this enzyme and of two enzymes with sequence similarity to ACLY. The first is succinyl-CoA synthetase (SCS), which catalyzes the formation of succinyl-CoA from succinate and CoA using ATP (4). The second is citrate synthase, the enzyme that generates citrate from acetyl-CoA and oxaloacetate in mitochondria. The reaction catalyzed by ACLY is thought to occur in four steps (Reactions 1-4):where E represents ACLY. Like SCS, ACLY is phosphorylated by ATP on an active site histidine residue to give E-P in step 1 (Reaction 1) (5, 6). The phosphoryl group is transferred to citrate in step 2 (Reaction 2). Citryl-phosphate is thought to remain bound to the enzyme, symbolized by E⅐citryl-P. Phosphate is released in the attack by CoA to form the citryl-CoA thioester bond in step 3 (Reaction 3) (7). In the last step (Reaction 4), citryl-CoA is cleaved to give acetyl-CoA and oxaloacetate, the reverse reaction to that catalyzed by citrate synthase.

Cullin3–KLHL25 ubiquitin ligase targets ACLY for degradation to inhibit lipid synthesis and tumor progression

“…1I). These two domains have been reported to be crucial for ACLY activity (Sun et al 2010). These results together demonstrate that ACLY directly interacts with KLHL25 to form a complex with CUL3 in cells.…”

Structure of NDP-forming Acetyl-CoA synthetase ACD1 reveals a large rearrangement for phosphoryl transfer

“…A comparable mode of interaction of the Ac group of Ac-CoA interacting with ACLY or ckcACD1, respectively, might be assumed. Hence, we superimposed ckcACD1 onto the crystal structure of human ACLY with bound citrate (17). The resulting distance between the carbonyl carbon of Ac-CoA of ckcACD1 and the carbon C1 of citrate in ACLY is ∼2.8 Å.…”

“…This phenomenon, termed "domain shuffling," is one of the key features of this superfamily (8). Superposition of several structures of SCS from Escherichia coli (ecSCS) (12)(13)(14), Thermus aquaticus (15), the mammalian GTP-specific SCS from pig (16), and a truncated form of human ACLY (17,18) revealed that subdomains 1-5 share a common arrangement in these enzymes. From detailed studies of the reaction mechanism of ecSCS, a crucial enzyme tightly connected to the TCA cycle, a three-step mechanism was proposed, which involves the phosphorylation of a highly conserved His residue at the first active site (site I) as an intermediate step (12,13).…”

Association of genetic variations in the ACLY gene with growth traits in Chinese beef cattle

“…NP_001087), residues 2-425 form domains 3, 4, and 5, and all three of these domains are homologous to the β subunit of succinyl-CoA synthetase. Residues 487-820 form domains 1 and 2, which are homologous to the α subunit of succinyl-CoA synthetase (Sun et al, 2010). In the present study, two SNPs g.17127C>T and g.35520C>T identified in bovine ACLY were found to be located in domains 5 and 2 of human ACLY isoform 1, respectively.…”

“…In mammals, ACLY is a homotetramer, containing an ATP-grasp domain (N-terminal region), a CoA binding domain, a CoA-ligase, and citrate synthetase (C-terminal region) (Morita et al, 2014). Sun et al (2010Sun et al ( , 2011 successfully identified the citrate and ATP binding site of chymotrypsin-truncated human ACLY using X-ray crystallography, revealing the partial residue conformation of the 1101-amino acid protein.…”