The overexpression of the ARD gene in E. coli yields two enzymes with distinct actions that are in a position to be separated by traditional chromatographic methods of ion trade and hydrophobic conversation resins. One particular enzyme catalyzes the onpathway oxidation of acireductone to ketoacid and formate the other enzyme catalyzes an off-pathway oxidation with development of CO. The only variation between the two enzymes (Fe-ARD and NiARD) was the steel certain in the lively internet sites [58]. There is structural info available for ARD from Mus musculus (PDB code: 1vr3) [sixty one] and Klebsiella pneumonia (PDB code: one zrr) (Figures 1G and 2F) [sixty two]. In the a lot more permissive structure network (Figure 3A) the two ARD constructions, like the RmlC epimerases, are linked to the greater cluster by means of dTDP-6-deoxy-3,4-keto-hexulose (PDB code: 2pa7) [sixty three] but shed all edges in the more stringent network (Determine 3B). Equally, in the much more permissive sequence community (Figure 6A) the ARD structures share an edge with every single other as effectively as other proteins but partition off into an impartial doublet (sharing a one edge) beneath a much more stringent threshold (Determine 6B). That users of the cupin superfamily cluster by operate when the operate is outlined at a fine stage allows 1 to make useful inferences which may supply a beginning stage for biochemical investigations. For case in point, closest neighbor examination of the protein item of AT3G21360 of Arabidopsis thaliana (PBD code: 1y0z) [sixty four] in the far more stringent structure community (Figure 3B, not shown with arrows) suggests that the protein item of AT3G21360 might be an alpha Integrin Antagonist 1 (hydrochloride) ketoglutarate-dependent TauD. This inference is more supported by the exclusive partitioning of this protein with PDB codes “3v15” and “10s7” (both confirmed TauDs) in the a 
lot more stringent domain-dependent network (Determine 4B). Similarly, the uncharacterized Escherichia coli protein Yr (PDB code: 3bb6) shares an edge exclusively with the Vibrio fisheri tellurite resistance protein B (PDB code: 3dl3) in both the a lot more stringent composition community (Determine 3B) and the considerably less stringent area-based mostly community (Figure 4A) (not proven with arrows in both community).
The Jumonji C area-made up of proteins are a subfamily of the Fe(II)/two-oxoglutarate-dependent oxygenases. Sequence similarity between JmjC and cupin metalloenzyme domains permitted the prediction of energetic-site residues in JmjC domains and offered the insights that guided the experimental perseverance of the reactions catalyzed by these enzymes [65]. The JmjC area-made up of factor inhibiting hypoxia (FIH) (Figures 1I and 2H) was recognized as an asparaginyl hydroxylase that transcriptionally regulates the exercise of 12909200hypoxia-inducible issue (HIF) [sixty six]. JmjC domaincontaining proteins had been proposed to purpose as histone demethylases in regulating chromatin construction [67] and a lot of of the JmjC area-made up of enzymes have been demonstrated to comprise the greatest class of histone demethylases that catalyze lysine demethylation of mono-, di-, and trimethylated lysine residues by means of the development of a hemiaminal intermediate that yields the demethylated merchandise and formaldehyde [68,69]. The lysine demethylases are represented by lysine-certain demethylase 6B in Figures 1J, 2I, and 2J. Moreover, JmjC domain-containing enzymes have been identified that have RNA hydroxylase action [21,70]. Most (twelve) of these are given the Pfam family members designation `JmjC’.
