Structural basis for recruitment of tandem hotdog domains in acyl-CoA thioesterase 7 and its role in inflammation.
Acyl-CoA thioesterases (Acots) catalyze the hydrolysis of fatty acyl-CoA to free fatty acid and CoA and thereby regulate lipid metabolism and cellular signaling. We present a comprehensive structural and functional characterization of mouse acyl-CoA thioesterase 7 (Acot7). Whereas prokaryotic homologues possess a single thioesterase domain, mammalian Acot7 contains a pair of domains in tandem. We determined the crystal structures of both the N- and C-terminal domains of the mouse enzyme, and inferred the structure of the full-length enzyme using a combination of chemical cross-linking, mass spectrometry, and molecular modeling. The quaternary arrangement in Acot7 features a trimer of hotdog fold dimers. Both domains of Acot7 are required for activity, but only one of two possible active sites in the dimer is functional. Asn-24 and Asp-213 (from N- and C-domains, respectively) were identified as the catalytic residues through site-directed mutagenesis. An enzyme with higher activity than wild-type Acot7 was obtained by mutating the residues in the nonfunctional active site. Recombinant Acot7 was shown to have the highest activity toward arachidonoyl-CoA, suggesting a function in eicosanoid metabolism. In line with the proposal, Acot7 was shown to be highly expressed in macrophages and up-regulated by lipopolysaccharide. Overexpression of Acot7 in a macrophage cell line modified the production of prostaglandins D2 and E2. Together, the results link the molecular and cellular functions of Acot7 and identify the enzyme as a candidate drug target in inflammatory disease.
Forwood
Jade K
JK
School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland 4072, Australia. jforwood@csu.edu.au
Thakur
Anil S
AS
Guncar
Gregor
G
Marfori
Mary
M
Mouradov
Dmitri
D
Meng
Weining
W
Robinson
Jodie
J
Huber
Thomas
T
Kellie
Stuart
S
Martin
Jennifer L
JL
Hume
David A
DA
Kobe
Bostjan
B
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D019281:Dimerization; D005456:Fluorescent Dyes; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects; D007211:Indoles; D007249:Inflammation; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000201:enzymology / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010170:Palmitoyl-CoA Hydrolase / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D015854:Up-Regulation / Q000187:drug effects
D005456:Fluorescent Dyes; D007211:Indoles; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; C007293:DAPI; C521939:Acot7 protein, mouse; D010170:Palmitoyl-CoA Hydrolase