Fig. 4: Phylogenetic (a) and structural (b) modeling of W-dependent oxidoreductases, and a metabolic model (c) illustrating the proposed link between cellular W usage and energy metabolism in W. gerlachensis.

a Maximum-likelihood phylogenetic tree of W-dependent ferredoxin oxidoreductase protein sequences retrieved from MAGs belonging to Wolframiiraptoraceae, and reference sequences. Illustrated is the masked alignment of the W-dependent ferredoxin oxidoreductase sequences, excluding all alignment positions with sequence information in less than 75% of the taxa analyzed. Branch support was inferred with SH-aLRT and Ultrafast bootstrapping and support at deeper nodes among the lineages in the phylogeny is indicated. W-dependent ferredoxin oxidoreductase lineages were inferred based on the topologies of the masked and full alignments (Supplementary Fig. 7 and Supplementary Data 7). The scale bar indicates the number of amino acid changes per site. b Multidimensional scaling of modeled protein structures. Data points with the most similar structural neighborhoods are positioned near each other. Protein models are based on the amino acid sequences aligned in a. Reference sequences are color-coded. K-means clustering was applied to identify significant data clusters (colored ellipses) based on minimum cluster r². Clusters included AORs from both references and those from representative members of the genus Wolframiiraptor. One sequence (MCF3653595.1) was excluded from the analysis (<300 amino acids). Dotted lines are used to indicate putative groups identified in a and b. All AOR-like lineages grouped together in Clusters I and II, while GAPOR-like sequences grouped in Cluster III and FOR-like sequences grouped in Cluster IV. c Pyruvate, derived from xylose via the pentose phosphate pathway (PPP) and the Embden–Meyerhof pathway (EMP), is stepwise converted to organic acids. Within this conversion, AOR and GAPOR couple aldehyde oxidation with ferredoxin reduction. Electrons from ferredoxin are used by a membrane-bound proton-translocating [NiFe]-hydrogenase (Mbh) to generate a proton motive force. Besides this main pathway, aldehydes could additionally be derived from amino acid metabolism and ferredoxin could also be recycled by an H₂-consuming sulfide dehydrogenase⁸⁹ after H₂ activation by Mbh (dashed lines). W uptake protein (subunits A-C), Tup; Oligopeptide transporter, Opp; Aromatic aminotransferase or branched-chain amino acid aminotransferase, Ar/Bcat; 2-Ketoisovalerate ferredoxin:oxidoreductase, VOR; Maltose/maltodextrin transporter, Mal; Xylose isomerase, Xyl; Xylulose kinase, Xk; Ribose 5-phosphate isomerase, Rpi; Transketolase, Tk; Coenzyme A (-acyl), CoASH; Aldehyde:ferredoxin oxidoreductase, AOR; Ion translocator, Mrp; sulfide dehydrogenase, SuDH; F0F1(V)-type ATP synthase, ATPase.