Invariant Thr244 is essential for the efficient acylation step of the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans

Abstract

One of the most striking features of several X-ray structures of CoA-independent ALDHs (aldehyde dehydrogenases) in complex with NAD(P) is the conformational flexibility of the NMN moiety. However, the fact that the rate of the acylation step is high in GAPN (non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase) from Streptococcus mutans implies an optimal positioning of the nicotinamide ring relative to the hemithioacetal intermediate within the ternary GAPN complex to allow an efficient and stereospecific hydride transfer. Substitutions of serine for invariant Thr244 and alanine for Lys178 result in a drastic decrease of the efficiency of hydride transfer which becomes rate-limiting. The crystal structure of the binary complex T244S GAPN-NADP shows that the absence of the beta-methyl group leads to a well-defined conformation of the NMN part, including the nicotinamide ring, clearly different from that depicted to be suitable for an efficient hydride transfer in the wild-type. The approximately 0.6-unit increase in pK(app) of the catalytic Cys302 observed in the ternary complex for both mutated GAPNs is likely to be due to a slight difference in positioning of the nicotinamide ring relative to Cys302 with respect to the wild-type ternary complex. Taken together, the data support a critical role of the Thr244 beta-methyl group, held in position through a hydrogen-bond interaction between the Thr244 beta-hydroxy group and the epsilon-amino group of Lys178, in permitting the nicotinamide ring to adopt a conformation suitable for an efficient hydride transfer during the acylation step for all the members of the CoA-independent ALDH family.

Adenine Binding Mode Is a Key Factor in Triggering the Early Release of NADH in Coenzyme A-dependent Methylmalonate Semialdehyde Dehydrogenase

“…The side chain of invariant Glu-399 was shown to play an essential role in this stabilization by anchoring the NMN ribose through hydrogen bonds with its hydroxyl groups (19). Moreover, recent studies support a critical contribution of the ␤-methyl group of the invariant Thr-244 residue that allows the nicotinamide ring to adopt a productive conformation for hydride transfer (20). Nevertheless, this conformation is not suitable for the deacylation because it would sterically preclude the catalytic Glu-268 from playing its role in the hydrolytic process.…”

Catalytic contribution of threonine 244 in human ALDH2

“…Similarly, we have shown that the T244S in human ALDH1A1 resulted in decreased catalytic efficiency and increased K M for NAD + [29]. In both cases, the binding of NADH was only slightly impaired [19,29]. Finally, we have also reported that substitution of Thr-244 reduces catalytic efficiency in human ALDH3A2 as well [30].…”

“…1A). The coenzyme NAD(P) + binds to the first Rossmann fold in an unusual manner, where the adenosine occupies a cleft between the αF and αG helices, the pyrophosphates are solvent exposed, and the nicotinamide mononucleotide has been observed to occupy various positions [16–19] (Fig. 1B).…”

“…Threonine 244 is one highly conserved residue whose function has only recently been examined in detail [19,29,30]. X-ray crystal structures show that Thr-244 lies between the coenzyme- and substrate-binding sites with the beta-methyl positioned to interact with the B-face of the nicotinamide ring, when coenzyme is positioned for hydride transfer (Fig.…”

“…2) [17,18]. Recently, Pailot, et al showed that substitution of Thr-244 in non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) resulted in decreased catalytic efficiency, increased K M for cofactor and a small shift in the position of the nicotinamide ring (Fig 1B, 2id2) [19]. Similarly, we have shown that the T244S in human ALDH1A1 resulted in decreased catalytic efficiency and increased K M for NAD + [29].…”