Crystal structure of NAD+-dependent Peptoniphilus asaccharolyticus glutamate dehydrogenase reveals determinants of cofactor specificity

Structure of NADP+‐dependent glutamate dehydrogenase from Escherichia coli – reflections on the basis of coenzyme specificity in the family of glutamate dehydrogenases

“…Protomer F (EcGDH-F) is the only molecule in the hexamer that adopts an open conformation that is sufficiently wide for diffusion of both substrate and cofactor. In contrast to EcGDH, the structures of all other wild-type GDHs in the substrate-free state have an open conformation [5, 7, 19, 23]. The distance between the basic residue (Lys136) in Domain I and the 2’-phosphate in the model of EcGDH/NADP + is only 7.6Å (not shown).…”

“…This aspartate is the equivalent of Glu243 in PaGDH (P7 position). In PaGDH, the glutamate forms hydrogen bonds with the 2’OH and 3’OH of adenine ribose, thus pointing in a completely different direction [19]. The subsequent residue Ser264, which we propose to designate P8, makes a hydrogen bond to the 2’-phosphate of NADP + (3.4Å).…”

“…6, green). However, the proximal lysine (Lys268) is further away from the 2’-phosphate (4.5Å) upon modeling of NADP + into the active site of PaGDH [19] relative to EcGDH, in which the proximal lysine is significantly closer to the 2’-phosphate. PaGDH also lacks the basic residue in Domain I (equivalent of Lys136 of EcGDH) that may contribute to NADP + stabilization in the fully closed and catalytically competent conformation.…”

“…However, CsGDH is an exception that violates the fingerprint rules [12, 17], differing in the residue at P6 (Ala) and P7 (Gly) despite rigorous (20,000-fold) NAD + discrimination over NADP + [18]. In marked contrast, our recent structure of another bacterial NAD + -dependent GDH, that of Peptoniphilus asaccharolyticus (PaGDH), reveals a pattern much more in keeping with the conventional view described above; a glutamate at the P7 position (Glu243) hydrogen bonds to 2’OH and 3’OH of the adenosine ribose facilitated by other residues in the vicinity that allow close approach to the sugar [19]. The comparison of these two structures thus bears out the view that a single enzyme family may offer strikingly different ways of achieving the same specificity.…”

BdcA, a Protein Important for Escherichia coli Biofilm Dispersal, Is a Short-Chain Dehydrogenase/Reductase that Binds Specifically to NADPH

“…Interestingly, the observed K d for BdcA with NADPH complex is ∼7–15 times higher than that of other members in the immediate SDR family (SDRvv:NADPH, K d  = 3.5 µM; ZmRDH:NAD, K d  = 2.72 µM; DHDPR:NADPH, K d  = 1.5 µM) [35]–[37]. However, the binding affinity is comparable with other oxidoreductases that are more distantly related (PaGDH:NADH, K d  = 18.5 µM; OcDH:NADH: K d  = 14 µM) [38], [39]. Titration of NADP into BdcA exhibited an isotherm indicative of even weaker binding ( Figure 3C ) with titration of NAD, NADH and c-di-GMP into BdcA resulting in only heats of dilution; i.e., no binding ( Figure 3D ).…”