An N-terminal fragment of mouse DGAT1 binds different acyl-CoAs with varying affinity.
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 (cisDelta(13)22:1)-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.
Siloto
Rodrigo M P
RM
Department of Agricultural, Food and Nutritional Science, 4-10 Agriculture/Forestry Centre, University of Alberta, Edmonton, Alta., Canada T6G 2P5.
Madhavji
Milan
M
Wiehler
William B
WB
Burton
Tracy L
TL
Boora
Parveen S
PS
Laroche
André
A
Weselake
Randall J
RJ
D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D029688:Brassica napus / Q000201:enzymology / Q000235:genetics; D017124:Conserved Sequence; D003911:Dextrans / Q000737:chemistry; D051048:Diacylglycerol O-Acyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000214:Acyl Coenzyme A; D003911:Dextrans; D011994:Recombinant Proteins; C010599:lipidex; C494882:Dgat1 protein, mouse; D051048:Diacylglycerol O-Acyltransferase