Pailot Arnaud, D'Ambrosio Katia, Corbier Catherine, Talfournier François, Branlant Guy
MAEM, UMR 7567 Nancy-Université, CNRS, Faculté des Sciences, 54506 Vandoeuvre Cedex, France.
Biochem J. 2006 Dec 15;400(3):521-30. doi: 10.1042/BJ20060843.
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.
与NAD(P)结合的不依赖辅酶A的醛脱氢酶（ALDHs）的几种X射线结构中，最显著的特征之一是NMN部分的构象灵活性。然而，变形链球菌的非磷酸化甘油醛-3-磷酸脱氢酶（GAPN）中酰化步骤速率很高这一事实表明，在三元GAPN复合物中，烟酰胺环相对于半硫代乙缩醛中间体处于最佳位置，以实现高效且立体特异性的氢化物转移。将不变的苏氨酸244替换为丝氨酸，以及将赖氨酸178替换为丙氨酸，会导致氢化物转移效率急剧下降，该步骤成为限速步骤。二元复合物T244S GAPN-NADP的晶体结构表明，β-甲基的缺失导致NMN部分（包括烟酰胺环）具有明确的构象，这与被描述为适合野生型中高效氢化物转移的构象明显不同。在两种突变的GAPN的三元复合物中观察到催化性半胱氨酸302的pK(app)大约增加0.6个单位，这可能是由于相对于野生型三元复合物，烟酰胺环相对于半胱氨酸302的定位略有不同。综上所述，数据支持苏氨酸244的β-甲基基团通过苏氨酸244的β-羟基与赖氨酸178的ε-氨基之间的氢键相互作用保持在特定位置，在使烟酰胺环采用适合不依赖辅酶A的ALDH家族所有成员在酰化步骤中进行高效氢化物转移的构象方面发挥关键作用。
