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. 2021 Jul 15;6(9):1129–1139. doi: 10.1038/s41564-021-00934-8

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 1 — The sequence of Kuenenia stuttgartiensis Kustd1700 (WP_099324707.1) was aligned to its orthologues from Jettenia caeni (Planctomycete KSU-1, KSU1_B0257, WP_007220240.1), Brocadia fulgida (BROFUL_02528, KKO18624.1) and Scalindua brodae (SCABRO_01076, KHE93157.1) using ClustalO⁵⁰. The alignment also contains sequences of the NXR A subunits from the nitrite-oxidizing bacteria (NOB, including comammox species) Nitrospira defluvii (WP_013249749.1), Nitrospira nitrificans (WP_090900831.1), Nitrospira inopinata (WP_062483509.1) and Nitrospina gracilis (WP_042250442.1). Moreover, the sequences from Candidatus Nitrotoga sp. (MBA0901970.1), the hydrogen-oxidizing bacterium Hydrogenobaculum sp. Y04AAS1 (WP_012513384.1) and the nitrate reductase from Beggiatoa sp. 4572_84 (OQY53866.1) were aligned. The last group in the alignment comprises the NAR from Thiocapsa sp. KS1 (WP_093186287.1) as well as the NXR from Nitrolancea hollandica Lb (AFN37206.1) and the nitrate reductases from the Nitrobacter-type organisms Nitrobacter winogradskyi (WP_011315305.1), Nitrobacter hamburgensis (WP_041359063.1), Nitrococcus mobilis (WP_004998773.1) which are related to the dissimilatory nitrate reductase from Escherichia coli str. K-12 (NP_415742.1) as well as the alpha-subunit of ethylbenzene dehydrogenase (EbdA) from Aromatoleum aromaticum (Q5P5I0). K. stuttgartiensis NXR A shares about 85% sequence identity with its orthologues from J. caeni and B. fulgida as well as 66% with S. brodae. The sequence identities are around 60% with the NOB and comammox homologues 40% with Nitrotoga and Hydrogenobaculum, whereas they are only around 20% with the Nitrobacter-type enzymes, the nitrate reductases and EbdA. Fully conserved peptide sequences are marked black. The residues binding to the [4Fe-4S] cluster (FS0) are highlighted in red and marked with a red triangle (C67, D71, C75, C115). The predicted tat signal peptides are highlighted in grey, and the proposed substrate-coordinating N70, which is I in ethylbenzene dehydrogenase, is shown in light blue. The figure was prepared using ESPript⁵¹. The complete alignment is provided as a supplemental file.