Abstract

6-Phosphogluconate dehydrogenases (6PGDHs) catalyze a key oxidative step in the oxidative pentose phosphate pathway (oxPPP), a route essential for NAD(P)H generation and carbon metabolism in bacteria and eukaryotes. While the structural basis of substrate recognition is well established for long-chain dimeric 6PGDHs, the mechanisms used by short-chain tetrameric enzymes remain poorly defined. Here, we present a 2.0 & Aring; crystal structure of tetrameric 6PGDH from Gluconobacter oxydans (Go6PGDH) in complex with 6-phosphogluconate (6PG) and integrate it with evolutionary, computational, and functional analyses. The structure shows that, unlike dimeric homologs, tetrameric Go6PGDH does not undergo a domain-closure transition upon ligand binding. Instead, 6PG induces a compaction of the tetramer mediated by two conserved C-terminal elements: an inter-protomer ionic "lock" and an intra-subunit C-terminal "latch" that together stabilize a closed catalytic pocket. Molecular-dynamics simulations identify His328 as a central residue that couples C-terminal tail closure to direct ligand coordination, and mutagenesis analysis confirms its essential role in catalytic efficiency. Thermodynamic measurements reveal that 6PG binding is strongly enthalpy-driven, consistent with the formation of an ordered hydrogen-bonding and electrostatic network in the closed conformation. These findings define a substrate-induced quaternary-tightening mechanism unique to tetrameric 6PGDHs and illustrate how a conserved C-terminal module has been adapted across the family to regulate substrate binding and catalysis.