The role of histidine 200 in MndD, the Mn(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase from Arthrobacter globiformis CM-2, a site-directed mutagenesis study
The role of histidine 200 in MndD, the Mn(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase from Arthrobacter globiformis CM-2, a site-directed mutagenesis study
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DOI：
10.1007/s00775-005-0017-1
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发表时间：
2005-11-01
影响因子：
3
通讯作者：
Wackett, LP
中科院分区：
文献类型：
--
作者：
Emerson, JP;Wagner, ML;Wackett, LP
来自球形节杆菌 CM-2 的锰依赖性 3,4-二羟基苯乙酸酯 2,3-双加氧酶 (MndD) 是一种额外二醇裂解儿茶酚双加氧酶，可催化 3,4-二羟基苯乙酸酯 (DHPA) 的芳环裂解。基于 MndD-DHPA 复合物的最新晶体结构，对保守的第二球残基组氨酸 200 进行了一系列定点突变，以深入了解并阐明该残基在 Mn(II) 依赖性催化机制中所起的作用。在这项研究中，我们报告了这些 H200 变体及其 DHPA 和 4-硝基儿茶酚 (4-NC) 复合物的活性和光谱数据。从野生型和突变型 MndD 收集的数据与 H200 与锰结合双氧部分相互作用的作用一致，并且与先前提出的涉及质子转移的其他作用不一致。讨论了光谱观察结果，包括当 DHPA 和 4-NC 与 MndD 的 Mn(II) 中心结合时发现的独特的低场 EPR 信号，并探讨了它们与 MndD 中催化的分子氧活化的关系。
The manganese-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase (MndD) from Arthrobacter globiformis CM-2 is an extradiol-cleaving catechol dioxygenase that catalyzes aromatic ring cleavage of 3,4-dihydroxyphenylacetate (DHPA). Based on the recent crystal structure of the MndD-DHPA complex, a series of site-directed mutations were made at a conserved second-sphere residue, histidine 200, to gain insight into and clarify the role this residue plays in the Mn(II)-dependent catalytic mechanism. In this study, we report the activities and spectroscopic data of these H200 variants and their DHPA and 4-nitrocatechol (4-NC) complexes. The data collected from wild-type and mutant MndDs are consistent with a role for H200 interacting with a manganese-bound dioxygen moiety and are inconsistent with other previously proposed roles involving proton transfer. Spectroscopic observations, including unique low-field EPR signals found when DHPA and 4-NC are bound to the Mn(II) center of MndD, are discussed and their relationship to dioxygen activation catalyzed in MndD is explored.