A Conserved Seven Amino Acid Stretch Important for Murine Mitochondrial Glycerol-3-phosphate Acyltransferase Activity

Abstract

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and committed step in glycerolipid biosynthesis. We previously cloned the cDNA sequence to murine mitochondrial GPAT (Yet, S-F., Lee, S., Hahm, Y. T., and Sul, H.S. (1993) Biochemistry 32, 9486 -9491). We expressed the protein in insect cells which was targeted to mitochondria, purified, and reconstituted mitochondrial GPAT activity using phospholipids (Yet, S.-F., Moon, Y., and Sul, H. S. (1995) Biochemistry 34, 7303-7310). Deletion of the seven amino acids from mitochondrial GPAT, 312 IFLEGTR 318 , which is highly conserved among acyltransferases in glycerolipid biosynthesis, drastically reduced mitochondrial GPAT activity. Treatment of mitochondrial GPAT with arginine-modifying agents, phenylglyoxal and cyclohexanedione, inactivated the enzyme. Two highly conserved arginine residues, Arg-318, in the seven amino stretch, and Arg-278, were identified. Substitution of Arg-318 with either alanine, histidine, or lysine reduced the mitochondrial GPAT activity by over 90%. On the other hand, although substitution of Arg-278 with alanine and histidine decreased mitochondrial GPAT activity by 90%, replacement with lysine reduced activity by only 25%. A substitution of the nonconserved Arg-279 with either alanine, histidine, or lysine did not alter mitochondrial GPAT activity. Moreover, R278K mitochondrial GPAT still showed sensitivity to arginine-modifying agents, as in the case of wild-type mitochondrial GPAT. These results suggest that Arg-318 may be critical for mitochondrial GPAT activity, whereas Arg-278 can be replaced by a basic amino acid. Examination of the other conserved residues in the seven amino acid stretch revealed that Phe-313 and Glu-315 are also important, but conservative substitutions can partially maintain activity; substitution with alanine reduced activity by 83 and 72%, respectively, whereas substituting Phe-313 with tyrosine and Glu-315 with glutamine had even lesser effect. In addition, there was no change in fatty acyl-CoA selectivity. Kinetic analysis of the R318K and R318A mitochondrial GPAT showed an 89 and 95%, respectively, decrease in catalytic efficiency but no major change in substrate binding as indicated by the K m values for palmitoyl-CoA and glycerol 3-phosphate. These studies indicate importance of the conserved seven amino acid stretch for mitochondrial GPAT activity and the significance of Arg-318 for catalysis.

Cloning and functional characterization of a novel mitochondrial N-ethylmaleimide-sensitive glycerol-3-phosphate acyltransferase (GPAT2)

“…Using the 798 GPAT2 amino acid sequence, the Blast alignment of GPAT1 and GPAT2 showed an amino acid sequence identity of 27% with GPAT1 and, in the active site region from amino acid 153 to 372, an amino acid identity of 24.5%. The active site region contained recognizable motifs 1 and 4 that had been identified in E. coli PlsB [8,14] and mouse GPAT1 [15], and that are present in dihydroxyacetonephosphate acyltransferase [1], lysocardiolipin acyltransferase [16], and lysophosphatidylglycerol acyltransferase [17]. Motifs 2 and 3, however, differed in GPAT2.…”

“…In mammals and bacteria, motif 2 normally contains the sequence FXXR, and the invariant arginine is essential for the activity of human tafazzin, human dihydroxyacetonephosphate acyltransferase and E. coli PlsB, probably because it interacts with the phosphate portion of the glycerolphosphate substrates of these enzymes [1]. Motif 3 usually contains the consensus sequence FXEGXRXR in which the glutamic acid, glycine, and the first arginine are essential for activity [1,15]. In GPAT2, however, the usual motif 2 and 3 sequences differ markedly from GPAT1 and are similar, instead, to sequences in several chloroplast GPATs (Fig.…”

Triacylglycerol Metabolism In Adipose Tissue

“…Our laboratory originally cloned mtGPAT, which was the first mammalian enzyme in TAG and phospholipid biosynthesis to be cloned [15][16][17]. While mammalian cells are known to have two GPATs, one in the ER (microsomal GPAT) and another in the mitochondria (mtGPAT), microsomal GPAT has not been cloned [13,18].…”

“…Most enzymes in TAG synthesis are intrinsic membrane proteins and their substrates and products are hydrophobic, making purification and kinetic analysis difficult. Cloning and characterization of the enzymes mentioned previously has only been achieved during the last decade following advances in molecular biological techniques.Our laboratory originally cloned mtGPAT, which was the first mammalian enzyme in TAG and phospholipid biosynthesis to be cloned [15][16][17]. While mammalian cells are known to have two GPATs, one in the ER (microsomal GPAT) and another in the mitochondria (mtGPAT), microsomal GPAT has not been cloned [13,18].…”

Mitochondrial Glycerol Phosphate Acyltransferase Contains Two Transmembrane Domains with the Active Site in the N-terminal Domain Facing the Cytosol

“…Recombinant mouse (3) and rat (4) GPAT open reading frames encode proteins of 827 and 828 amino acids, respectively, with a high degree of homology. Alignment of rat mitochondrial GPAT with other acyltransferases shows four homology blocks (5), and when conserved amino acids within these regions are mutated, GPAT activity is altered or abolished (5)(6)(7)(8). Kinetic studies using recombinant GPAT with site-directed mutations in this region identify specific amino acids that contribute to substrate binding or to catalysis.…”