School of Biological Sciences, University of East Anglia, Norwich, United Kingdom.
School of Environmental Sciences, University of East Anglia, Norwich, United Kingdom.
Appl Environ Microbiol. 2020 Apr 17;86(9). doi: 10.1128/AEM.02388-19.
Ammonia monooxygenase (AMO) is a key nitrogen-transforming enzyme belonging to the same copper-dependent membrane monooxygenase family (CuMMO) as the particulate methane monooxygenase (pMMO). The AMO from ammonia-oxidizing archaea (AOA) is very divergent from both the AMO of ammonia-oxidizing bacteria (AOB) and the pMMO from methanotrophs, and little is known about the structure or substrate range of the archaeal AMO. This study compares inhibition by C to C linear 1-alkynes of AMO from two phylogenetically distinct strains of AOA, " Nitrosocosmicus franklandus" C13 and " Nitrosotalea sinensis" Nd2, with AMO from and pMMO from (Bath). An increased sensitivity of the archaeal AMO to short-chain-length alkynes (≤C) appeared to be conserved across AOA lineages. Similarities in C to C alkyne inhibition profiles between AMO from AOA and pMMO from suggested that the archaeal AMO has a narrower substrate range than AMO. Inhibition of AMO from " Nitrosocosmicus franklandus" and by the aromatic alkyne phenylacetylene was also investigated. Kinetic data revealed that the mechanisms by which phenylacetylene inhibits " Nitrosocosmicus franklandus" and are different, indicating differences in the AMO active site between AOA and AOB. Phenylacetylene was found to be a specific and irreversible inhibitor of AMO from " Nitrosocosmicus franklandus," and it does not compete with NH for binding at the active site. Archaeal and bacterial ammonia oxidizers (AOA and AOB, respectively) initiate nitrification by oxidizing ammonia to hydroxylamine, a reaction catalyzed by ammonia monooxygenase (AMO). AMO enzyme is difficult to purify in its active form, and its structure and biochemistry remain largely unexplored. The bacterial AMO and the closely related particulate methane monooxygenase (pMMO) have a broad range of hydrocarbon cooxidation substrates. This study provides insights into the AMO of previously unstudied archaeal genera, by comparing the response of the archaeal AMO, a bacterial AMO, and pMMO to inhibition by linear 1-alkynes and the aromatic alkyne, phenylacetylene. Reduced sensitivity to inhibition by larger alkynes suggests that the archaeal AMO has a narrower hydrocarbon substrate range than the bacterial AMO, as previously reported for other genera of AOA. Phenylacetylene inhibited the archaeal and bacterial AMOs at different thresholds and by different mechanisms of inhibition, highlighting structural differences between the two forms of monooxygenase.
氨单加氧酶（AMO）是一种关键的氮转化酶，属于与颗粒态甲烷单加氧酶（pMMO）相同的铜依赖性膜单加氧酶家族（CuMMO）。氨氧化古菌（AOA）的 AMO 与氨氧化细菌（AOB）的 AMO 和产甲烷菌的 pMMO 非常不同，对于古菌 AMO 的结构或底物范围知之甚少。本研究比较了两种系统发育上不同的 AOA 菌株“ Nitrosocosmicus franklandus” C13 和“ Nitrosotalea sinensis” Nd2 的 AMO 以及 中的 AMO 和 pMMO 对 C 到 C 直链 1-炔烃的抑制作用。古菌 AMO 对短链长度炔烃（≤C）的敏感性增加似乎在 AOA 谱系中得到了保守。AOA 的 AMO 与 中的 pMMO 之间 C 到 C 炔烃抑制谱的相似性表明，古菌 AMO 的底物范围比 中的 AMO 更窄。芳香炔烃苯乙炔对“ Nitrosocosmicus franklandus”的 AMO 和 的抑制作用也进行了研究。动力学数据表明，苯乙炔抑制“ Nitrosocosmicus franklandus”和 的机制不同，表明 AOA 和 AOB 之间 AMO 活性位点存在差异。发现苯乙炔是“ Nitrosocosmicus franklandus” AMO 的特异性和不可逆抑制剂，它不会与活性位点的 NH 竞争结合。氨氧化古菌（AOA）和氨氧化细菌（AOB）分别通过将氨氧化为羟胺来启动硝化作用，这一反应由氨单加氧酶（AMO）催化。AMO 酶难以以其活性形式纯化，其结构和生物化学仍在很大程度上未被探索。细菌 AMO 和密切相关的颗粒态甲烷单加氧酶（pMMO）具有广泛的烃共氧化底物。本研究通过比较未研究的古菌属的 AMO、细菌 AMO 和 pMMO 对直链 1-炔烃和芳香炔烃苯乙炔的抑制反应，为以前未研究的古菌属的 AMO 提供了一些见解。对较大炔烃抑制的敏感性降低表明，与其他 AOA 属先前报道的情况一样，古菌 AMO 的烃底物范围比细菌 AMO 窄。苯乙炔以不同的阈值抑制古菌和细菌 AMO，并通过不同的抑制机制，突出了两种单加氧酶之间的结构差异。
