The effect of methane and methanol on the terrestrial ammonia‐oxidizing archaeon ‘Candidatus Nitrosocosmicus franklandus C13’

Abstract

The ammonia monooxygenase (AMO) is a key enzyme in ammonia‐oxidizing archaea, which are abundant and ubiquitous in soil environments. The AMO belongs to the copper‐containing membrane monooxygenase (CuMMO) enzyme superfamily, which also contains particulate methane monooxygenase (pMMO). Enzymes in the CuMMO superfamily are promiscuous, which results in co‐oxidation of alternative substrates. The phylogenetic and structural similarity between the pMMO and the archaeal AMO is well‐established, but there is surprisingly little information on the influence of methane and methanol on the archaeal AMO and terrestrial nitrification. The aim of this study was to examine the effects of methane and methanol on the soil ammonia‐oxidizing archaeon ‘Candidatus Nitrosocosmicus franklandus C13’. We demonstrate that both methane and methanol are competitive inhibitors of the archaeal AMO. The inhibition constants (Ki) for methane and methanol were 2.2 and 20 μM, respectively, concentrations which are environmentally relevant and orders of magnitude lower than those previously reported for ammonia‐oxidizing bacteria. Furthermore, we demonstrate that a specific suite of proteins is upregulated and downregulated in ‘Ca. Nitrosocosmicus franklandus C13’ in the presence of methane or methanol, which provides a foundation for future studies into metabolism of one‐carbon (C1) compounds in ammonia‐oxidizing archaea.

Quantifying Microbial Guilds

“…Thus, the ancestral AMO enzyme has shifted from a moderate affinity for a broad spectrum of substrates to a restricted substrate specificity, suboptimal for the substrate preference of each organism [43]. Therefore, other closely related enzymes retained some promiscuity or ambivalence; for example, particulate methane monooxygenases (pMMO) are able to oxidize ammonia even though their main substrate is methane [44, 45], or pBMO, which, apart from oxidizing butane, can hydroxylate other short alkanes (C 1 -C 9 ) [46, 47]. Therefore, we can discriminate between proteins carrying out ammonia oxidation with u = 1 and others that have higher affinities for methane or other simple aliphatic alkanes [48], with u ∼ 0 for ammonia oxidation (Fig.…”

“…Thus, the ancestral AMO enzyme has shifted from a moderate affinity for a broad spectrum of substrates to a restricted substrate specificity, suboptimal for the substrate preference of each organism [43]. Therefore, other closely related enzymes retained some promiscuity or ambivalence; for example, particulate methane monooxygenases (pMMO) are able to oxidize ammonia even though their main substrate is methane [44,45],…”

Alcohols as inhibitors of ammonia oxidizing archaea and bacteria

“…N. franklandus’ C13, which equate to ∼1.2 and 5.55 µg protein ml −1 , respectively. Cell counts were performed as previously described (Oudova-Rivera et al 2023 ). Protein concentrations were determined using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific) according to the manufacturer instructions.…”

“…Interestingly, phenylacetylene and octyne do not interact with the same site of the archaeal AMO as ammonia and acetylene, and AOA are inhibited by phenylacetylene and octyne at higher concentrations than AOB (Wright et al 2020 ). Methane is a competitive inhibitor of the AMO in both archaea and bacteria (Suzuki et al 1976 , Oudova-Rivera et al 2023 ). Oxidation of alkanes and alkenes by CuMMO superfamily monooxygenases, including the bacterial AMO, generates alcohols as reaction products (Hyman et al 1988b ).…”

“…Recently, methanol was shown to be a specific competitive inhibitor of the archaeal AMO from ‘ Ca . N. franklandus’ C13 (Oudova-Rivera et al 2023 ). Furthermore, specific inhibition of the bacterial AMO from N. europaea by short-chain linear C 1 -C 4 alcohols, including methanol, ethanol, and both isomers of propanol and butanol, was previously reported (Hooper and Terry 1973 ).…”