Cloning and expression of a human ATP‐citrate lyase cDNA

Abstract

A full‐length cDNA clone of 4.3 kb encoding the human ATP‐citrate lyase enzyme has been isolated by screening a human cDNA library with the recently isolated rat ATP‐citrate lyase cDNA clone [Elshourbagy et al. (1990) J. Biol. Chem. 265, 1430]. Nucleic‐acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1105 amino acids in length with a calculated molecular mass of 121419 Da. Comparison of the human and rat ATP‐citrate lyase cDNA sequences reveals 96.3% amino acid identity throughout the entire sequence. Further sequence analysis identified the His765 catalytic phosphorylation site, the ATP‐binding site, as well as the CoA binding site. The human ATP‐citrate lyase cDNA clone was subcloned into a mammalian expression vector for expression in African green monkey kidney cells (COS) and Chinese hamster ovary cells (CHO) cells. Transfected COS cells expressed detectable levels of an enzymatically active recombinant ATP‐citrate lyase enzyme. Stable, amplified expression of ATP‐citrate lyase in CHO cells was achieved by using coamplification with dihydrofolate reductase. Resistant cells expressed high levels of enzymatically active ATP‐citrate lyase (3 pg/cell/d). Site‐specific mutagenesis of His765→Ala diminishes the catalytic activity of the expressed ATP‐citrate lyase protein. Since catalysis of ATP‐citrate lyase is postulated to involve the formation of phosphohistidine, these results are consistent with the pattern of earlier observations of the significance of the histidine residue in catalysis of the human ATP‐citrate lyase.

Cell Differentiation during Sexual Development of the Fungus Sordaria macrospora Requires ATP Citrate Lyase Activity

“…3]) of the ORF, and flanking sequences show the highest level of similarity with translation initiation sites from other S. macrospora genes (41). The putative polypeptide from S. macrospora has a calculated molecular mass of 73 kDa and thus has only about two-thirds of the molecular mass of animal ACL polypeptides (11,12). The S. macrospora ACL1 polypeptide has homology with the C-terminal part of the corresponding animal polypeptides, which carries the enzyme's proposed catalytic center, including the histidine residue that is autophosphorylated during the catalyzed reaction (Fig.…”

Pathogen‐induced expression of plant ATP:citrate lyase1

“…Calculated molecular mass of hot pepper ACL polypeptide was 66 kDa, which is only half of the animal ACLs. The ACL catalytic center, GHAGA, and the histidine residue that is autophosphorylated by ATP during catalysis are highly conserved in Ca-ACL1, but none of the three additional phosphorylation sites (Tyr446, Ser450, Ser454) that are involved in the regulation of rat ACL activity exists [2].To investigate the expression kinetics of the Ca-ACL1 transcripts during inoculation with X. campestris pv. glycines8ra, total RNA was isolated from hot pepper leaves 0, 4, 8, and 12 h after inoculation, and Northern blot analysis was performed by using 32 P-labeled Ca-ACL1 as a probe.…”

“…Calculated molecular mass of hot pepper ACL polypeptide was 66 kDa, which is only half of the animal ACLs. The ACL catalytic center, GHAGA, and the histidine residue that is autophosphorylated by ATP during catalysis are highly conserved in Ca-ACL1, but none of the three additional phosphorylation sites (Tyr446, Ser450, Ser454) that are involved in the regulation of rat ACL activity exists [2].…”

Identification and characterization of a mammalian 14‐kDa phosphohistidine phosphatase

“…One is the intermediary phosphorylation of enzymes [5][6][7][8][9][10], of which nucleoside diphosphate kinase is a particularly well-studied example. The other is the reversible protein histidine phosphorylation by protein kinases and phosphatases [3,11].…”