Lontoh S, DiSpirito A A, Krema C L, Whittaker M R, Hooper A B, Semrau J D
Department of Civil and Environmental Engineering, The University of Michigan, Ann Arbor 48109-2125, USA.
Environ Microbiol. 2000 Oct;2(5):485-94. doi: 10.1046/j.1462-2920.2000.00130.x.
Phenylacetylene was investigated as a differential inhibitor of ammonia monooxygenase (AMO), soluble methane monooxygenase (sMMO) and membrane-associated or particulate methane monooxygenase (pMMO) in vivo. At phenylacetylene concentrations > 1 microM, whole-cell AMO activity in Nitrosomonas europaea was completely inhibited. Phenylacetylene concentrations above 100 microM inhibited more than 90% of sMMO activity in Methylococcus capsulatus Bath and Methylosinus trichosporium OB3b. In contrast, activity of pMMO in M. trichosporium OB3b, M. capsulatus Bath, Methylomicrobium album BG8, Methylobacter marinus A45 and Methylomonas strain MN was still measurable at phenylacetylene concentrations up to 1,000 microM. AMO of Nitrosococcus oceanus has more sequence similarity to pMMO than to AMO of N. europaea. Correspondingly, AMO in N. oceanus was also measurable in the presence of 1,000 microM phenylacetylene. Measurement of oxygen uptake indicated that phenylacetylene acted as a specific and mechanistic-based inhibitor of whole-cell sMMO activity; inactivation of sMMO was irreversible, time dependent, first order and required catalytic turnover. Corresponding measurement of oxygen uptake in whole cells of methanotrophs expressing pMMO showed that pMMO activity was inhibited by phenylacetylene, but only if methane was already being oxidized, and then only at much higher concentrations of phenylacetylene and at lower rates compared with sMMO. As phenylacetylene has a high solubility and low volatility, it may prove to be useful for monitoring methanotrophic and nitrifying activity as well as identifying the form of MMO predominantly expressed in situ.
在体内研究了苯乙炔作为氨单加氧酶（AMO）、可溶性甲烷单加氧酶（sMMO）和膜结合或颗粒状甲烷单加氧酶（pMMO）的差异抑制剂。当苯乙炔浓度>1微摩尔时，欧洲亚硝化单胞菌中的全细胞AMO活性被完全抑制。高于100微摩尔的苯乙炔浓度抑制了荚膜甲基球菌巴斯菌株和嗜甲基孢囊菌OB3b中90%以上的sMMO活性。相比之下，在高达1000微摩尔的苯乙炔浓度下，嗜甲基孢囊菌OB3b、荚膜甲基球菌巴斯菌株、白色甲基微菌BG8、海栖甲基杆菌A45和甲基单胞菌菌株MN中的pMMO活性仍可测量。海洋亚硝化球菌的AMO与pMMO的序列相似性比与欧洲亚硝化单胞菌的AMO更高。相应地，在1000微摩尔苯乙炔存在的情况下，海洋亚硝化球菌中的AMO也可测量。氧气摄取测量表明，苯乙炔作为全细胞sMMO活性的特异性和基于机制的抑制剂；sMMO的失活是不可逆的、时间依赖性的、一级的，并且需要催化周转。对表达pMMO的甲烷营养菌全细胞中的氧气摄取进行相应测量表明，pMMO活性受到苯乙炔的抑制，但前提是甲烷已经在被氧化，而且只有在苯乙炔浓度高得多且与sMMO相比速率较低的情况下才会受到抑制。由于苯乙炔具有高溶解度和低挥发性，它可能被证明可用于监测甲烷营养和硝化活性以及识别原位主要表达的MMO形式。
