A sequence similarity network representation of the proteins in cog0402. A BLAST network diagram was constructed and rendered with Cytoscape (22). At an E-value cutoff of 10^-70 cog0402 separates into more than 10 groups, each thought to have a different function. These groups use different active site residues to bind substrates but share identical residues for catalysis and metal binding. The identities of the groups are as follows: 1: SAH deaminase, 2: guanine deaminase, 3: unknown, 4: 8-oxoguanine/isoxanthopterin deaminase, 5: unknown, 6: cytosine deaminase, 7: unknown, 8: N-formimino-L-glutamate deiminase, 9: unknown, and 10: unknown.

Absorption spectra of the reaction mixtures before and after the addition of purified cytosine deaminase to either isoguanine (top panel) or cytosine (bottom panel) at pH 7.7. Isoguanine and cytosine (spectra in blue) were incubated with 1.6 μM CDA for 1.5 hours in 45 mM HEPES, pH 7.7. After the reaction was complete, the UV spectra (shown in red) were collected. The red spectrum in the top panel is consistent with the formation of xanthine (λ_max = 272 nm) from isoguanine (λ_max = 286 nm). The red spectrum in the bottom panel is consistent with formation of uracil (λ_max = 260 nm) from cytosine (λ_max = 267 nm).

The structure of isoguanine (in green) bound in the active site of cytosine deaminase.

A sequence similarity network for cog0402 is presented in Figure 1 at an E-value cutoff of 10^-70 (21). We postulated that there may be a subset of enzymes within cog0402 that is able to deaminate isoguanine to xanthine as shown in Scheme 1. The most likely candidate for this activity was predicted to be in a group of enzymes related to S-adenosylhomocysteine deaminase in Group 1 of Figure 1. Group 1 is the largest and most diverse group of enzymes within cog0402. SAH deaminase utilizes a conserved histidine residue (His-137) to hydrogen bond with N3 of the adenine moiety of SAH for substrate recognition (17). This particular histidine residue is fully conserved across all Group 1 enzymes of cog0402 except for three small subgroups, one of which contains a glutamine at this position. It was initially hypothesized that the carboxamide moiety of the glutamine side chain could be positioned in the active site of these enzymes to hydrogen bond with the C2/N3 carbamoyl group of isoguanine. From this small subgroup of enzymes, an uncharacterized protein from Picrophilus torridus was selected for purification and characterization. Since the DNA from this extremophilic archaeon was not commercially available, we purchased the codon-optimized gene (gi∣48477797) from GenScript and then attempted to express the protein in an E. coli host.