Inhibition of Ammonia Monooxygenase from Ammonia-Oxidizing Archaea by Linear and Aromatic Alkynes

Abstract

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 C2 to C8 linear 1-alkynes of AMO from two phylogenetically distinct strains of AOA, “Candidatus Nitrosocosmicus franklandus” C13 and “Candidatus Nitrosotalea sinensis” Nd2, with AMO from Nitrosomonas europaea and pMMO from Methylococcus capsulatus (Bath). An increased sensitivity of the archaeal AMO to short-chain-length alkynes (≤C5) appeared to be conserved across AOA lineages. Similarities in C2 to C8 alkyne inhibition profiles between AMO from AOA and pMMO from M. capsulatus suggested that the archaeal AMO has a narrower substrate range than N. europaea AMO. Inhibition of AMO from “Ca. Nitrosocosmicus franklandus” and N. europaea by the aromatic alkyne phenylacetylene was also investigated. Kinetic data revealed that the mechanisms by which phenylacetylene inhibits “Ca. Nitrosocosmicus franklandus” and N. europaea 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 “Ca. Nitrosocosmicus franklandus,” and it does not compete with NH3 for binding at the active site. IMPORTANCE 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.

Comparison of Novel and Established Nitrification Inhibitors Relevant to Agriculture on Soil Ammonia- and Nitrite-Oxidizing Isolates

“…EQ, QI, EQNL, and NP were added to the cultures as filter sterilized dimethyl sulfoxide (DMSO) solutions due to their low water solubility (≤60 mg L –1 at 20°C). The final concentration of DMSO in all cultures was 0.1% (v/v), which did not exert a significant inhibitory effect to any of the isolates tested (data not shown), in line with previous studies with the same isolates ( Wright et al, 2020 ; Zhao et al, 2020 ). DCD and DMPP were dissolved in sterile dH 2 O before addition of 25 μl (0.5% v/v).…”

“…Hundreds of compounds have been identified that inhibit nitrifying prokaryotes ( Bédard and Knowles, 1989 ; McCarty, 1999 ) including plant-derived molecules ( Coskun et al, 2017 ), aliphatic and aromatic n-alkynes ( Taylor et al, 2015 ; Wright et al, 2020 ), statins ( Zhao et al, 2020 ), and PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide; Martens-Habbena et al, 2015 ). Many of these are used as selective inhibitors of ammonia-oxidizing bacteria (AOB; e.g., octyne) or archaea (AOA; e.g., PTIO) in laboratory cultures, soil microcosms or slurries, but are not suitable for use in an agricultural setting due to rapid degradation in soil or application in a gaseous state.…”

“…The use of inhibition assays with pure cultures of a diverse range of soil-derived strains is a necessary benchmarking step to define the exact spectrum of activity of NIs destined for use in agriculture. Most culture inhibition assays have focused on AOB (e.g., Bélser and Schmidt, 1981 ; Vannelli and Hooper, 1992 ) or tested NIs not broadly applied in agricultural settings on soil AOA strains (i.e., allylthiourea, n-aliphatic alkynes, and simvastatin; Wright et al, 2020 ; Zhao et al, 2020 ). Others have explored the activity of NIs of agricultural relevance on AOA ( Jung et al, 2011 ; Kim et al, 2012 ), but only three provided a systematic assessment and inhibition thresholds for AOA soil strains like “ Candidatus Nitrosocosmicus agrestis” ( Liu et al, 2019 ), “ Candidatus Nitosotalea devanaterra” ( Lehtovirta-Morley et al, 2013 ), and Nitrososphaera viennensis ( Shen et al, 2013 ).…”

An activity-based labelling method for the detection of ammonia and methane-oxidizing bacteria

“…The ability of 1,7OD to act as an irreversible and mechanism-based inactivator of the AMO enzyme in the canonical AOB Nitrosomonas europaea was previously demonstrated (19), and the pMMO of Methylococcus capsulatus (Bath) was shown to be partially inhibited by alkynes with chain lengths ≤C7 (94). However, comammox Nitrospira harbor a phylogenetically distinct AMO enzyme (1,2,36).…”

“…First, there appeared to be enrichment of nitrifying bacteria due to treatment steps prior to sorting, particularly among the sonicated and ethanol-treated samples, but also by the CuACC reaction itself (Figure 8). These effects may be due to their tendency to grow as microcolonies within biomass aggregates, which may protect them from the effects of sonication, ethanol fixation, and the CuAAC reaction (92)(93)(94). Second, the apparent most abundant ammonia-oxidizer after ABPP-based labelling and cell sorting depended on the marker gene, as a Nitrosomonas-like canonical AOB appeared to be most highly enriched based on amoA abundance increases, while this was a comammox Nitrospira in the rpoB analysis.…”