Redesign of Schistosoma mansoni NAD+ catabolizing enzyme: active site H103W mutation restores ADP-ribosyl cyclase activity.
Redesign of Schistosoma mansoni NAD+ catabolizing enzyme: active site H103W mutation restores ADP-ribosyl cyclase activity.
复制标题
曼氏血吸虫NAD分解代谢酶的重新设计：活性位点H103W突变恢复ADP-核糖基环化酶活性。
DOI：
10.1021/bi060930g
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发表时间：
2006
期刊：
影响因子：
2.9
通讯作者：
Schuber,Francis
中科院分区：
文献类型：
--
作者：
Kuhn,Isabelle;Kellenberger,Esther;Rognan,Didier;Lund,FrancesE;Muller-Steffner,Hélène;Schuber,Francis
曼氏血吸虫NAD（P）+分解代谢酶（SmNACE）是ADP-核糖环化酶家族的新成员。与所有其他参与产生引起Ca 2+动员的代谢物的酶相反，SmNACE几乎不能将NAD+转化为第二信使环ADP-核糖（cADPR）。序列比对显示，这些酶的活性位点内的4个保守残基之一被替换inSmNACE由组氨酸（His 103），而不是高度保守的色氨酸。为了查明SmNACE催化经典ADP-核糖基环化酶反应的能力是否与这种变化有关，我们用色氨酸取代了His 103。确实发现SmNACE中的H103 W突变恢复了ADP-核糖基环化酶活性，因为对于7%的反应产物，cADPR量（即，该值大于对该家族的其他成员如CD 38观察到的值）。Trp 103残基的引入提供了哺乳动物ADP-核糖基环化酶的一些结合特性，例如对Cibacron蓝的亲和力增加和araF-NAD+的缓慢结合抑制。野生型和H103 W突变体三维结构的同源建模，以及活性位点内底物的对接，为SmNACE的催化机制提供了新的见解。两个残基侧链在烟酰胺-核糖键裂解步骤中具有相似的作用，导致E. ADP-核糖基反应中间体。然而，它们在该中间体的演变中存在分歧; His 103提供了一个更极性的环境，有利于水的可及性和水解，从而以导致cADPR的分子内环化途径为代价产生ADP-核糖。
Schistosoma mansoniNAD(P)+catabolizing enzyme (SmNACE) is a new member of the ADP-ribosyl cyclase family. In contrast to all the other enzymes that are involved in the production of metabolites that elicit Ca2+mobilization,SmNACE is virtually unable to transform NAD+into the second messenger cyclic ADP-ribose (cADPR). Sequence alignments revealed that one of four conserved residues within the active site of these enzymes was replaced inSmNACE by a histidine (His103) instead of the highly conserved tryptophan. To find out whether the inability ofSmNACE to catalyze the canonical ADP-ribosyl cyclase reaction is linked to this change, we have replaced His103with a tryptophan. The H103W mutation inSmNACE was indeed found to restore ADP-ribosyl cyclase activity as cADPR amounts for 7% of the reaction products (i.e., a value larger than observed for other members of this family such as CD38). Introduction of a Trp103residue provides some of the binding characteristics of mammalian ADP-ribosyl cyclases such as increased affinity for Cibacron blue and slow-binding inhibition by araF-NAD+. Homology modeling of wild-type and H103W mutant three-dimensional structures, and docking of substrates within the active sites, provides new insight into the catalytic mechanism ofSmNACE. Both residue side chains share similar roles in the nicotinamide−ribose bond cleavage step leading to an E.ADP-ribosyl reaction intermediate. They diverge, however, in the evolution of this intermediate; His103provides a more polar environment favoring the accessibility to water and hydrolysis leading to ADP-ribose at the expense of the intramolecular cyclization pathway resulting in cADPR.