Novel Structures of Type 1 Glyceraldehyde-3-phosphate Dehydrogenase from Escherichia coli Provide New Insights into the Mechanism of Generation of 1,3-Bisphosphoglyceric Acid

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly conserved enzyme involved in the ubiquitous process of glycolysis and presents a loop (residues 208–215 of Escherichia coli GAPDH) in two alternative conformations (I and II). It is uncertain what triggers this loop rearrangement, as well as which is the precise site from which phosphate attacks the thioacyl intermediate precursor of 1,3-bisphosphoglycerate (BPG). To clarify these uncertainties, we determined the crystal structures of complexes of wild-type GAPDH (WT) with NAD and phosphate or G3P, and of essentially inactive GAPDH mutants (C150S, H177A), trapping crystal structures for the thioacyl intermediate or for ternary complexes with NAD and either phosphate, BPG, or G3P. Analysis of these structures reported here lead us to propose that phosphate is located in the “new Pi site” attacks the thioester bond of the thioacyl intermediate to generate 1,3-bisphosphoglyceric acid (BPG). In the structure of the thioacyl intermediate, the mobile loop is in conformation II in subunits O, P, and R, while both conformations coexist in subunit Q. Moreover, only the Q subunit hosts bound NADH. In the R subunit, only the pyrophosphate part of NADH is well defined, and NADH is totally absent from the O and P subunits. Thus, the change in loop conformation appears to occur after NADH is produced, before NADH is released. In addition, two new D-glyceraldehyde-3-phosphate (G3P) binding forms are observed in WT.NAD.G3P and C150A+H177A.NAD.G3P. In summary, this paper improves our understanding of the GAPDH catalytic mechanism, particularly regarding BPG formation.

Non-enzymatic acetylation inhibits glycolytic enzymes in Escherichia coli

“…The mechanism of inhibition for GpmA is most likely through direct interference of K100 acetylation with binding to its substrate phosphoglycerate. 38 Since K184 of GapA is not directly involved in catalysis or substrate binding, 39,40 we considered the catalytic cavity closest to K184 that anchors glyceraldehyde 3-phosphate by binding to threonine 180, threonine 182, and cysteine 150 (C150). We hypothesized that acetylation at K184 may sterically interfere with glyceraldehyde 3-phosphate binding.…”

Strategy to Select an Appropriate Cryoprotectant for an X-ray Study of Escherichia coli GAPDH Crystals

“…36,37 EcGAPDH has been crystallized with various precipitating agents, not only salts such as ammonium sulfate or sodium citrate but also polymers, e.g., PEG 400, 1000, 3350, 4000, and in a relatively wide pH range (4.5−8.5). 6,[28][29][30][31]33,34 The cryoprotectant trehalose was used when EcGAPDH was crystallized with either ammonium sulfate or sodium citrate. As a cryoprotectant itself, trehalose does not have negative effects on resolution, with the exception that bound trehalose induces a 2.4°rotation at the S-loop compared with the NAD +free EcGAPDH structure and a 3.1°interdomain rotation compared with the NAD + -bound EcGAPDH structure.…”

Structure-Guided Protein Engineering of Glyceraldehyde-3-phosphate Dehydrogenase from Corynebacterium glutamicum for Dual NAD/NADP Cofactor Specificity

“…GAPDH is an essential glycolytic enzyme that converts glyceraldehyde-3-phosphate (G3P) to 1,3-bisphophoglycerate, and it utilizes one molecule of NAD and an inorganic phosphate as cofactors (Figure A). Since the first crystal structure of GAPDH from Homarus americanus (lobster) was reported in 1975, a large number of GAPDH structures from various organisms, including bacteria, animal, plant, yeast, and parasites, have been determined to elucidate diverse biological functions. Moreover, this kind of structural information can help identify the detailed molecular mechanism of GAPDH in glycolysis .…”