Castro Lucila I, Hermsen Corinna, Schultz Joachim E, Linder Jürgen U
Abteilung Pharmazeutische Biochemie, Fakultät für Chemie und Pharmazie, Universität Tübingen, Germany.
FEBS J. 2005 Jun;272(12):3085-92. doi: 10.1111/j.1742-4658.2005.04722.x.
Class III adenylyl cyclases usually possess six highly conserved catalytic residues. Deviations in these canonical amino acids are observed in several putative adenylyl cyclase genes as apparent in several bacterial genomes. This suggests that a variety of catalytic mechanisms may actually exist. The gene Rv0386 from Mycobacterium tuberculosis codes for an adenylyl cyclase catalytic domain fused to an AAA-ATPase and a helix-turn-helix DNA-binding domain. In Rv0386, the standard substrate, adenine-defining lysine-aspartate couple is replaced by glutamine-asparagine. The recombinant adenylyl cyclase domain was active with a V(max) of 8 nmol cAMP.mg(-1).min(-1). Unusual for adenylyl cyclases, Rv0386 displayed 20% guanylyl cyclase side-activity with GTP as a substrate. Mutation of the glutamine-asparagine pair either to alanine residues or to the canonical lysine-aspartate consensus abolished activity. This argues for a novel mechanism of substrate selection which depends on two non-canonical residues. Data from individual and coordinated point mutations suggest a model for purine definition based on an amide switch related to that previously identified in cyclic nucleotide phosphodiesterases.
Ⅲ类腺苷酸环化酶通常具有六个高度保守的催化残基。在几个细菌基因组中,一些假定的腺苷酸环化酶基因中观察到这些典型氨基酸存在偏差。这表明实际上可能存在多种催化机制。结核分枝杆菌的Rv0386基因编码一个与AAA-ATP酶和一个螺旋-转角-螺旋DNA结合结构域融合的腺苷酸环化酶催化结构域。在Rv0386中,标准底物、定义腺嘌呤的赖氨酸-天冬氨酸对被谷氨酰胺-天冬酰胺取代。重组腺苷酸环化酶结构域具有活性,V(max)为8 nmol cAMP·mg⁻¹·min⁻¹。与腺苷酸环化酶不同的是,Rv0386以GTP为底物时显示出20%的鸟苷酸环化酶副活性。将谷氨酰胺-天冬酰胺对突变为丙氨酸残基或典型的赖氨酸-天冬氨酸共有序列会使活性丧失。这表明存在一种依赖于两个非典型残基的新型底物选择机制。来自单个和协同点突变的数据表明了一种基于与先前在环核苷酸磷酸二酯酶中鉴定的酰胺开关相关的嘌呤定义模型。