Structural and functional characterization of the intracellular filament-forming nitrite oxidoreductase multiprotein complex

Abstract

Nitrate is an abundant nutrient and electron acceptor throughout Earth’s biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the nitrite oxidoreductase (NXR) multiprotein complex. NXR is a crucial enzyme in the global biological nitrogen cycle, and is found in nitrite-oxidizing bacteria (including comammox organisms), which generate the bulk of the nitrate in the environment, and in anaerobic ammonium-oxidizing (anammox) bacteria which produce half of the dinitrogen gas in our atmosphere. However, despite its central role in biology and decades of intense study, no structural information on NXR is available. Here, we present a structural and biochemical analysis of the NXR from the anammox bacterium Kuenenia stuttgartiensis, integrating X-ray crystallography, cryo-electron tomography, helical reconstruction cryo-electron microscopy, interaction and reconstitution studies and enzyme kinetics. We find that NXR catalyses both nitrite oxidation and nitrate reduction, and show that in the cell, NXR is arranged in tubules several hundred nanometres long. We reveal the tubule architecture and show that tubule formation is induced by a previously unidentified, haem-containing subunit, NXR-T. The results also reveal unexpected features in the active site of the enzyme, an unusual cofactor coordination in the protein’s electron transport chain, and elucidate the electron transfer pathways within the complex.

Introducing Candidatus Bathyanammoxibiaceae, a family of bacteria with the anammox potential present in both marine and terrestrial environments

“…Scalinduaceae, members of Ca . Bathyanammoxibiaceae also encode the nitrite oxidoreductase (NXR), which in anammox bacteria is an essential enzyme with a tubular architecture functioning in the anammoxosome organelle [ 43 ]. Phylogenetic analysis of the nitrite oxidoreductase alpha subunit (NxrA) showed that members of Ca .…”

Some like it cold: the cellular organization and physiological limits of cold‐tolerant nitrite‐oxidizing Nitrotoga

“…In contrast to other NOB, these rows of particles in Nitrotoga are multilayered and further indicated a soluble nature of the NXR, as most of the stacked particles have no direct contact to the cytoplasmic membrane. Similarly, NXR particles in anammox bacteria of the genus Kuenenia appear to be organized in tubule‐like structures located in the anammoxosome of the cells, where the energy delivering reactions take place (de Almeida et al ., 2015; Chicano et al ., 2021). In analogy, we suggest that the periplasm of Nitrotoga is an extended ‘compartment’ for energy conservation, especially considering that the NXR in anaerobic ammonium‐oxidizing planctomycetes is more closely related to the periplasmic enzymes of Nitrotoga , Nitrospira and Nitrospina , than to the cytoplasm‐oriented NXR‐type of for instance Nitrobacter (Supplementary Fig.…”

Phylogenomic Evidence for the Origin of Obligate Anaerobic Anammox Bacteria Around the Great Oxidation Event

“…Previous studies investigated the roles of key genes driving anammox, including hzsCBA (hydrazine synthase) (Dietl et al 2015), hdh (hydrazine dehydrogenase), hao (hydroxylamine oxidoreductase) (Kartal, Maalcke et al 2011), and nxr (nitrite oxidoreductase) (Chicano et al 2021). In general, the vital enzymes encoded by these genes either are directly involved in anammox or participate in replenishing electrons to the cyclic electron flow (Kartal and Keltjens 2016;Hu et al 2019).…”