Cystathionine β-Synthase (CBS) Domain-containing Pyrophosphatase as a Target for Diadenosine Polyphosphates in Bacteria
Cystathionine β-Synthase (CBS) Domain-containing Pyrophosphatase as a Target for Diadenosine Polyphosphates in Bacteria
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DOI：
10.1074/jbc.m115.680272
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发表时间：
2015-11-13
影响因子：
4.8
通讯作者：
Baykov, Alexander A.
中科院分区：
文献类型：
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作者：
Anashkin, Viktor A.;Salminen, Anu;Baykov, Alexander A.
在众多含有成对调节性胱硫醚β-合酶 (CBS) 结构域的蛋白质中，家族 II 焦磷酸酶 (CBS-PPase) 的独特之处在于它们通常在 CBS 结构域之间含有一个额外的 DRTGG 结构域。腺嘌呤核苷酸以正向合作的方式与 CBS-PPase 中的 CBS 结构域结合，导致酶抑制（AMP 或 ADP）或激活（ATP）。在这里，我们展示了线性 P-1,P-n-二腺苷 5'-多磷酸盐（Ap(n)As，其中 n 是磷酸残基的数量）以纳摩尔亲和力与脱硫杆菌、诺氏梭菌和产气荚膜梭菌的含有 DRTGG 结构域的 CBS-PPase 结合，并将其活性分别提高了 30 倍、5 倍和 7 倍。 Ap(4)A、Ap(5)A 和 Ap(6)A 以非合作方式结合并具有类似的高亲和力与 CBS-PPase，而 Ap(3)A 以正合作方式结合且具有较低的亲和力，如单核苷酸。所有 Ap(n)As 都废除了 CBS-PPase 的动力学协同性（非迈克尔行为）。通过等温量热法测定的焓变和结合化学计量与 Ap(4)A 和 Ap(5)A 的 10 kcal/mol 核苷酸和 1 mol/mol 酶二聚体相似，但 Ap(3)A、AMP、ADP 和 ATP 的焓变和结合化学计量为 5.5 kcal/mol 和 2 mol/mol，表明两个核苷酸组的结合模式不同。相比之下，不含DRTGG结构域的Eggerthella lenta和Mooreella thermoaceta CBS-PPases不受Ap(n)As的影响，并且没有表现出焓变化，这表明DTRGG结构域对于Ap(n)A结合的重要性。这些发现表明 Ap(n)As 可以控制 CBS-PPase 活性，从而影响细菌中的焦磷酸水平和生物合成活性。
Among numerous proteins containing pairs of regulatory cystathionine beta-synthase (CBS) domains, family II pyrophosphatases (CBS-PPases) are unique in that they generally contain an additional DRTGG domain between the CBS domains. Adenine nucleotides bind to the CBS domains in CBS-PPases in a positively cooperative manner, resulting in enzyme inhibition (AMP or ADP) or activation (ATP). Here we show that linear P-1,P-n-diadenosine 5'-polyphosphates (Ap(n)As, where n is the number of phosphate residues) bind with nanomolar affinity to DRTGG domain-containing CBS-PPases of Desulfitobacterium hafniense, Clostridium novyi, and Clostridium perfringens and increase their activity up to 30-, 5-, and 7-fold, respectively. Ap(4)A, Ap(5)A, and Ap(6)A bound noncooperatively and with similarly high affinities to CBS-PPases, whereas Ap(3)A bound in a positively cooperative manner and with lower affinity, like mononucleotides. All Ap(n)As abolished kinetic cooperativity (non-Michaelian behavior) of CBS-PPases. The enthalpy change and binding stoichiometry, as determined by isothermal calorimetry, were similar to 10 kcal/mol nucleotide and 1 mol/mol enzyme dimer for Ap(4)A and Ap(5)A but 5.5 kcal/mol and 2 mol/mol for Ap(3)A, AMP, ADP, and ATP, suggesting different binding modes for the two nucleotide groups. In contrast, Eggerthella lenta and Moorella thermoacetica CBS-PPases, which contain noDRTGG domain, were not affected by Ap(n)As and showed no enthalpy change, indicating the importance of the DTRGG domain for Ap(n)A binding. These findings suggest that Ap(n)As can control CBS-PPase activity and hence affect pyrophosphate level and biosynthetic activity in bacteria.