Structure of glycerol-3-phosphate dehydrogenase, an essential monotopic membrane enzyme involved in respiration and metabolism

Abstract

Sn-glycerol-3-phosphate dehydrogenase (GlpD) is an essential membrane enzyme, functioning at the central junction of respiration, glycolysis, and phospholipid biosynthesis. Its critical role is indicated by the multitiered regulatory mechanisms that stringently controls its expression and function. Once expressed, GlpD activity is regulated through lipid-enzyme interactions in Escherichia coli. Here, we report seven previously undescribed structures of the fully active E. coli GlpD, up to 1.75 Å resolution. In addition to elucidating the structure of the native enzyme, we have determined the structures of GlpD complexed with substrate analogues phosphoenolpyruvate, glyceric acid 2-phosphate, glyceraldehyde-3-phosphate, and product, dihydroxyacetone phosphate. These structural results reveal conformational states of the enzyme, delineating the residues involved in substrate binding and catalysis at the glycerol-3-phosphate site. Two probable mechanisms for catalyzing the dehydrogenation of glycerol-3-phosphate are envisioned, based on the conformational states of the complexes. To further correlate catalytic dehydrogenation to respiration, we have additionally determined the structures of GlpD bound with ubiquinone analogues menadione and 2-n-heptyl-4-hydroxyquinoline N-oxide, identifying a hydrophobic plateau that is likely the ubiquinone-binding site. These structures illuminate probable mechanisms of catalysis and suggest how GlpD shuttles electrons into the respiratory pathway. Glycerol metabolism has been implicated in insulin signaling and perturbations in glycerol uptake and catabolism are linked to obesity in humans. Homologs of GlpD are found in practically all organisms, from prokaryotes to humans, with >45% consensus protein sequences, signifying that these structural results on the prokaryotic enzyme may be readily applied to the eukaryotic GlpD enzymes.electron transfer ͉ glycerol metabolism ͉ glyceroneogenesis ͉ ubiquinone

Massive Formation of Intracellular Membrane Vesicles in Escherichia coli by a Monotopic Membrane-bound Lipid Glycosyltransferase

“…Lys and Arg are also common in the interface regions of typical monotopic membrane proteins, e.g. prostaglandin H2 synthase-1 (56), signal peptide peptidase (61), and glycerol-3-phosphate dehydrogenase (62). Of the three monotopic proteins here with very similar three-dimensional structures (i.e.…”

A Refined Analysis of Superoxide Production by Mitochondrial sn-Glycerol 3-Phosphate Dehydrogenase

“…Crystal structures of the homologous GlpD enzyme from Escherichia coli suggest that the FAD (substrate binding region) resides in the mitochondrial intermembrane space, whereas the Q-binding site sits in the outer leaflet (16). Both vertebrate and invertebrate mGPDH contain a calcium-binding EF-hand domain in the intermembrane space that lowers the K m for glycerol 3-phosphate at physiological levels of free calcium (17,18).…”

Crystal structure of the flavoenzyme PA4991 fromPseudomonas aeruginosa

“…The structure was therefore determined by molecular replacement with Phaser-MR (Bunkó czi et al, 2013). Initially, coordinates of putative homologues with PDB codes 2q6u (Carrell et al, 2007) and 2r4j (Yeh et al, 2008), both displaying less than 18% sequence identity to PA4991, were obtained from the PDB. Search models were derived from these coordinates by removing side chains and loop regions differing between the two structures, but no molecular-replacement solutions were found.…”

“…of 3.3 Å , 16% sequence identity and a Z-score of 26.6 (Tsuge et al, 2005), E. coli glycerol-3-phosphate dehydrogenase with an r.m.s.d. of 3.4 Å , 14% sequence identity and a Z-score of 26.4 (Yeh et al, 2008), glycine oxidase from Bacillus subtilis with an r.m.s.d. of 3.5 Å , 14% sequence identity and a Z-score of 26.2 (Settembre et al, 2003;Mö rtl et al, 2004), the -subunit of sarcosine oxidase from Corynebacterium sp.…”

“…Accessibility of the cofactor in PA4991 appears in part to be controlled by the loop comprising residues 54-67, which is disordered in PA4991. Crystal structures of related enzymes with bound ligands, for instance glycerol-3-phosphate dehydrogenase with bound dihydroxyacetone phosphate (Yeh et al, 2008), show that the corresponding loops fold over the active site and sequester the bound ligand. In the vicinity of the isoalloxazine ring there are the side chains of His53, Arg316 and Lys345 that potentially could be involved in ligand binding and/or catalysis.…”