Substrate recognition and selectivity of plant glycerol-3-phosphate acyltransferases (GPATs) fromCucurbita moscataandSpinacea oleracea

Abstract

Stromal glycerol-3-phosphate acyltransferases (GPAT) are responsible for the selective incorporation of saturated and unsaturated fatty-acyl chains into chloroplast membranes, which is an important determinant of a plant's ability to tolerate chilling temperatures. The molecular mechanisms of plant chilling tolerance were elucidated by creating chimeric GPATs between squash (Cucurbita moscata, chilling-sensitive) and spinach (Spinacea oleracea, chilling-tolerant) and the results were interpreted using structural information on squash GPAT determined by X-ray crystallography at 1.55 A Ê resolution. Enzymatic analysis of the chimeric GPATs showed that the chimeric GPATs containing the spinach region from residues 128 to 187 prefer the 18:1 unsaturated fatty acid rather than 16:0 saturated fatty acid. Structure analysis suggests that the size and character of the cavity that is formed from this region determines the speci®c recognition of acyl chains.

Functional characterization of human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 9: cloning, tissue distribution, gene structure, and enzymatic activity

“…Similar, strongly hydrophobic region is obvious in the AGPAT9 as well (Fig. 8); suggesting that these hydrophobic residues might similarly fold to form a hydrophobic tunnel to accommodate the acyl chain, as observed with the tertiary structure of squash (Cucurbita moschata) chloroplast gpat (Turnbull et al 2001a,b, Slabas et al 2002, Tamada et al 2004. Analysis of the predicted secondary structure of gpat and AGPATs show that the positioning of the HX 4 D domain is in the loop region between a beta strand (b c ) and alpha helix Figure 8 Partial protein sequence of human AGPAT2 and AGPAT9, and squash (Cucurbita moschata) chloroplast gpat showing the predicted secondary structure: Based on gpat crystal structure, the region between residues 91 and 270 was selected, which includes the enzyme's active site, NHX 4 D. Based on sequence homology between various isoforms of AGPAT1 through AGPAT9 (data not shown), protein region between amino acids, 61 and 270, were selected for secondary structure prediction of AGPAT2 and AGPAT9.…”

Homodimeric cross-over structure of the human granulocyte colony-stimulating factor (GCSF) receptor signaling complex

“…Initial phase information for hGCSF and CRH domain of hGCSF-R was obtained by molecular replacement (MR) with PHASER (27) using the coordinates of the 1:1 complex of hGCSF and CRH domain of mouse GCSF-R (Protein Data Bank ID code 1CD9). Because electron densities calculated using the initial phase information were insufficient for chain tracing of the Ig-like domain of hGCSF-R, derivatives were obtained by soaking crystals in 1 mM thimerosal or by expression of hGCSF in medium containing selenomethioine using a nonauxotroph strain for methionine (wild strain) (28). Data of derivatives were collected under absorption peak wavelength based on fluorescence profiles.…”

Human 1-Acylglycerol-3-phosphate O-Acyltransferase Isoforms 1 and 2

“…Flanking the other side of the entrance to the active site is a cluster of highly conserved positive residues that have been implicated in GPAT to the binding of the phosphate in glycerol 3-phosphate (37). These residues are highly conserved in both AGPAT1 and AGPAT2.…”

“…Three-dimensional Models for Human AGPAT1 and AGPAT2 Proteins-Because there is not yet a crystal structure available for AGPAT1 and AGPAT2, the structure of both proteins were modeled based on the available x-ray crystal structure of GPAT (37,46). Both modeled proteins preserve the basic folding of GPAT.…”

“…The PSIPRED protein structure prediction server was used to predict secondary structure of AGPAT proteins (34), transmembrane domains (35), and protein fold recognition based upon the crystal structure of known proteins (36). Three-dimensional models of human AGPAT1 (accession number: NP_006402.1) and AGPAT2 (accession number: CAH71722.1) were built using the crystal structure of glycerol-3-phosphate acyltransferase (GPAT1) from Cucurbita moscata as a template (Protein Data Bank code 1IUQ) (37). The modeling was performed using AMBER 9 (38) and PyMol (PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC).…”