Crystal structure of the 6-phosphogluconate dehydrogenase from Gluconobacter oxydans reveals tetrameric 6PGDHs as the crucial intermediate in the evolution of structure and cofactor preference in the 6PGDH family

Abstract

Background: The enzyme 6-phosphogluconate dehydrogenase (6PGDH) is the central enzyme of the oxidative pentose phosphate pathway. Members of the 6PGDH family belong to different classes: either homodimeric enzymes assembled from long-chain subunits or homotetrameric ones assembled from short-chain subunits. Dimeric 6PGDHs bear an internal duplication absent in tetrameric 6PGDHs and distant homologues of the β-hydroxyacid dehydrogenase (βHADH) superfamily. Methods: We use X-ray crystallography to determine the structure of the apo form of the 6PGDH from Gluconobacter oxydans (Go6PGDH). We carried out a structural and phylogenetic analysis of short and long-chain 6PGDHs. We put forward an evolutionary hypothesis explaining the differences seen in oligomeric state vs. dinucleotide preference of the 6PGDH family. We determined the cofactor preference of Go6PGDH at different 6-phosphogluconate concentrations, characterizing the wild-type enzyme and three-point mutants of residues in the cofactor binding site of Go6PGDH. Results: The structural comparison suggests that the 6PG binding site initially evolved by exchanging C-terminal α-helices between subunits. An internal duplication event changed the quaternary structure of the enzyme from a tetrameric to a dimeric arrangement. The phylogenetic analysis suggests that 6PGDHs have spread from Bacteria to Archaea and Eukarya on multiple occasions by lateral gene transfer. Sequence motifs consistent with NAD+- and NADP+-specificity are found in the β2-α2 loop of dimeric and tetrameric 6PGDHs. Site-directed mutagenesis of Go6PGDH inspired by this analysis fully reverses dinucleotide preference. One of the mutants we engineered has the highest efficiency and specificity for NAD+ so far described for a 6PGDH. Conclusions: The family 6PGDH comprises dimeric and tetrameric members whose active sites are conformed by a C-terminal α-helix contributed from adjacent subunits. Dimeric 6PGDHs have evolved from the duplication-fusion of the tetrameric C-terminal domain before independent transitions of cofactor specificity. Changes in the conserved β2-α2 loop are crucial to modulate the cofactor specificity in Go6PGDH.

Structural and Kinetic Insights Into the Molecular Basis of Salt Tolerance of the Short-Chain Glucose-6-Phosphate Dehydrogenase From Haloferax volcanii

“…Until recently, it was generally assumed that the oxidative branch of the pentose-phosphate pathway was absent in most of the archaeal organisms studied since homologs of classical bacterial G6PDH could not be identified in any archaeon, and a modified version of this pathway was proposed (Soderberg, 2005). Nevertheless, to date, the presence of G6PDH as well as 6-phosphogluconate dehydrogenase has been reported in Haloabacteria, Pacearchaeota, Thaumarchaeota, and Asgarchaeota organisms (Maturana et al, 2021), although SDR-G6PDH seems to be restricted to Halobacteria, as has been shown previously by Pickl and Schönheit (2015).…”

6-Phosphogluconate dehydrogenase and its crystal structures

“…It is now recognized that there are both homodimeric and homotetrameric 6PGDHs. The latter group are mainly found in prokaryotes, although a dimer-tetramer equilibrium affected by ligands has been reported for 6PGDH from the protist Trypanosoma brucei (Tsai & Chen, 1998;Hanau et al, 2013;Maturana et al, 2021;Sarmiento-Pavı ´a et al, 2021).…”

“…Some bacterial 6PGDHs are specific for NAD + , while others can use both NAD + and NADP + (Maturana et al, 2021). In the thermostable 6PGDH from the hyperthermophilic bacterium Thermotoga maritima the coenzyme preference could be reversed from NADP + to NAD + by site-directed topical reviews Acta Cryst.…”

“…5 and 8; Adams et al, 1994). Mutagenesis of the conserved arginine and asparagine in Lactococcus lactis and Gluconobacter oxydans 6PGDH demonstrated their role in specificity for NADP + over NAD + , while showing that aspartate, which is present in NAD + -dehydrogenases in place of the asparagine, hinders placement of the 2 0 -phosphate (Tetaud et al, 1999;Maturana et al, 2021). Among the residues in the coenzyme-binding site, Lys76 (Lys75 in the sheep structure; Fig.…”

“…Some key residues such as Ser128, Gly129 and Gly130, Lys183 and Asn187 have also been recognized to be highly conserved in dehydrogenases such as HIBADH (Ser124, Gly124 and Gly125, Lys173 and Asn177 in the rat sequence), which revealed that the -hydroxyacid dehydrogenase (-HADH) superfamily, including the HIBADH group and the PGDH group, has no requirement for divalent metal ions (Hawes et al, 1996;Lokanath et al, 2005;Park et al, 2016). The lysine and asparagine are inside the so-called catalytic motif identified in the superfamily structural tree (Maturana et al, 2021) Binding modes of NADPH (left; PDB entry 1pgo) and nicotinamide-8-bromoadenine dinucleotide phosphate (Nbr 8 ADP, an analogue of NADP + ; right; PDB entry 1pgn) to ovine 6PGDH. One sulfate is present in the substrate-binding site on the left while a pyrophosphate is on the right, as indicated by arrows (created using PyMOL).…”