Crystal structures of a bacterial 6‐phosphogluconate dehydrogenase reveal aspects of specificity, mechanism and mode of inhibition by analogues of high‐energy reaction intermediates

Abstract

Crystal structures of recombinant Lactococcus lactis 6‐phosphogluconate dehydrogenase (LlPDH) in complex with substrate, cofactor, product and inhibitors have been determined. LlPDH shares significant sequence identity with the enzymes from sheep liver and the protozoan parasite Trypanosoma brucei for which structures have been reported. Comparisons indicate that the key residues in the active site are highly conserved, as are the interactions with the cofactor and the product ribulose 5‐phosphate. However, there are differences in the conformation of the substrate 6‐phosphogluconate which may reflect distinct states relevant to catalysis. Analysis of the complex formed with the potent inhibitor 4‐phospho‐d‐erythronohydroxamic acid, suggests that this molecule does indeed mimic the high‐energy intermediate state that it was designed to. The analysis also identified, as a contaminant by‐product of the inhibitor synthesis, 4‐phospho‐d‐erythronamide, which binds in similar fashion. LlPDH can now serve as a model system for structure‐based inhibitor design targeting the enzyme from Trypanosoma species.

Conformational changes associated with cofactor/substrate binding of 6-phosphogluconate dehydrogenase from Escherichia coli and Klebsiella pneumoniae: Implications for enzyme mechanism

“…In the complex of 6PGDH and NADP + from Ll6PGDH/NADP + (Sundaramoorthy et al, 2007) and the Oa6PGDH/NADPH (Adams et al, 1994) binary complex, the cofactors are bound by both the coenzyme-binding domain and the helix domain. Superposition of coenzyme-binding sites from the crystal structures of Ec6PGDH/6PG/ATR, Oa6PGDH and Ll6PGDH provides a dynamic picture of how residues may change conformation during ligand binding (Fig.…”

“…It is noteworthy that none of the triad residues is bound to the Nam ring of Nbr 8 ADP in Oa6PGDH/Nbr 8 ADP binary complex and these different binding modes of the oxidized and reduced cofactors infer that NADPH is involved in the orientation of substrate and facilitating decarboxylation 3-keto 6PG (Cervellati et al, 2008;. In addition, structural and thermodynamic studies suggested a modulation of the conformational change for a single subunit of the homodimer preceding catalysis (Montin et al, 2007;Sundaramoorthy et al, 2007). When it complexed with the product or inhibitors, a difference of cofactor binding in the coenzymebinding domain of the two subunits in the homodimeric 6PGDH was observed (Sundaramoorthy et al, 2007), which suggested the half-of-the-sites enzyme mechanism (Montin et al, 2007).…”

“…In addition, structural and thermodynamic studies suggested a modulation of the conformational change for a single subunit of the homodimer preceding catalysis (Montin et al, 2007;Sundaramoorthy et al, 2007). When it complexed with the product or inhibitors, a difference of cofactor binding in the coenzymebinding domain of the two subunits in the homodimeric 6PGDH was observed (Sundaramoorthy et al, 2007), which suggested the half-of-the-sites enzyme mechanism (Montin et al, 2007). However, until recently, it is unclear why occupancy of one active site has an influence on the other.…”

“…Previously, K183 and E190 had been identified as the general base and general acid (Adams et al, 1994;Karsten et al, 1998;Zhang et al, 1999), respectively. The structures of 6PGDH from Ovis arries (sheep) liver (Oa6PGDH Adams et al, 1994), Trypanosoma brucei (Tb6PGDH Phillips et al, 1998), Lactococcus lactis (Ll6PGDH; (Sundaramoorthy et al, 2007), Saccharomyces cerevisiae (yeast; Sc6PGDH; (He et al, 2007) have been determined, and they have provided better understanding of the enzyme's function. Several key residues in the active sites of Ll6PGDH in complex with NADP + and Ru5P (PDB ID: 2IYP) are highly conserved, and the cofactor and substrate share similar binding modes with those in Oa6PGDH (Adams et al, 1994;Sundaramoorthy et al, 2007).…”

The 6-Phosphogluconate Dehydrogenase of Leishmania (Leishmania) mexicana: Gene Characterization and Protein Structure Prediction

“…The 6-Phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) is the third enzyme of the oxidative branch of the PPP and has been considered an appropriate target for drugs in trypanosomatids [Barrett and Gilbert, 2002;Hanau et al, 2004;Igoillo-Esteve et al, 2007]. Crystal structures of the 6PGDH enzyme from Trypanosoma brucei [Phillips et al, 1998] and Lactococcus lactis [Sundaramoorthy et al, 2007] have been elucidated and structural differences have been found in comparison with the mammalian enzymes. A variety of highly selective inhibitors of the enzyme of T. brucei has been described .…”