An N-terminal fragment of mouse DGAT1 binds different acyl-CoAs with varying affinity

• Published in: Biochemical and biophysical research communications Vol. 373; no. 3; pp. 350 - 354
• Main Authors: Siloto, Rodrigo M.P., Madhavji, Milan, Wiehler, William B., Burton, Tracy L., Boora, Parveen S., Laroche, André, Weselake, Randall J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 29.08.2008
• Subjects: Acyl Coenzyme A - chemistry; Acyl Coenzyme A - metabolism; Acyl-CoA binding; Allosteric modulation; Amino Acid Sequence; Animals; Binding Sites; Brassica napus; Brassica napus - enzymology; Brassica napus - genetics; Conserved Sequence; Dextrans - chemistry; Diacylglycerol acyltransferase; Diacylglycerol O-Acyltransferase - chemistry; Diacylglycerol O-Acyltransferase - genetics; Diacylglycerol O-Acyltransferase - metabolism; Escherichia coli; Escherichia coli - genetics; Lipidex-1000; Mice; Molecular Sequence Data; Mus musculus; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Alignment; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Mus musculus; Diacylglycerol acyltransferase; Lipidex-1000; Allosteric modulation; Acyl-CoA binding
• ISSN: 0006-291X; 1090-2104; 1090-2104
• DOI: 10.1016/j.bbrc.2008.06.031

A histidine-tagged recombinant N-terminal fragment of type-1 mouse liver diacylglycerol acyltransferase (DGAT; EC 2.3.1.20), MmDGAT1 (1–95)His6, was expressed in Escherichia coli, and used to investigate possible acyl-CoA-binding properties. Analysis of the purified fragment by MALDI-TOF mass spectrometry revealed a polypeptide with molecular mass of about 11 kDa which was consistent with the calculated molecular mass based on the deduced amino acid sequence. Lipidex-1000 binding assays indicated that MmDGAT1 (1–95)His 6 interacted with long chain fatty acyl-CoAs similar to observations on DGAT1 from oilseed rape ( Brassica napus). Binding, as a function of acyl-CoA concentration, differed for palmitoyl (16:0), stearoyl (18:0), and erucoyl ( cisΔ 1322:1)-CoA. Binding of stearoyl- or erucoyl-CoA to MmDGAT1 (1–95)His 6 as a function of acyl-CoA concentration, however, was sigmoid and displayed positive cooperativity suggesting that MmDGAT1 may be subject to allosteric modulation by acyl-CoAs. An intra-polypeptide segment within the N-terminal region of MmDGAT1 contained remnants of an acyl-CoA-binding signature initially identified in plant DGAT1. The acyl-CoA-binding site in mammalian DGAT1 could represent a potential target for therapeutic interventions for disorders such as type-2 diabetes and obesity.