The tautomerase superfamily (TSF) consists of more than 11,000 nonredundant sequences present throughout the biosphere. Characterized members have attracted much attention because of the unusual and key catalytic role of an N-terminal proline. These few characterized members catalyze a diverse range of chemical reactions, but the full scale of their chemical capabilities and biological functions remains unknown. To gain new insight into TSF structure–function relationships, we performed a global analysis of similarities across the entire superfamily and computed a sequence similarity network to guide classification into distinct subgroups. Our results indicate that TSF members are found in all domains of life, with most being present in bacteria. The eukaryotic members of the cis-3-chloroacrylic acid dehalogenase subgroup are limited to fungal species, whereas the macrophage migration inhibitory factor subgroup has wide eukaryotic representation (including mammals). Unexpectedly, we found that 346 TSF sequences lack Pro-1, of which 85% are present in the malonate semialdehyde decarboxylase subgroup. The computed network also enabled the identification of similarity paths, namely sequences that link functionally diverse subgroups and exhibit transitional structural features that may help explain reaction divergence. A structure-guided comparison of these linker proteins identified conserved transitions between them, and kinetic analysis paralleled these observations. Phylogenetic reconstruction of the linker set was consistent with these findings. Our results also suggest that contemporary TSF members may have evolved from a short 4-oxalocrotonate tautomerase–like ancestor followed by gene duplication and fusion. Our new linker-guided strategy can be used to enrich the discovery of sequence/structure/function transitions in other enzyme superfamilies.

The existence of cobalamin (Cbl)-dependent enzymes that are members of the radical S-adenosyl-L-methionine (SAM) superfamily was previously predicted based on bioinformatic analysis. A number of these are Cbl-dependent methyltransferases but the details surrounding their reaction mechanisms have remained unclear. In this report we demonstrate the in vitro activity of GenK, a Cbl-dependent radical SAM enzyme that methylates an unactivated sp3 carbon during the biosynthesis of gentamicin, an aminoglycoside antibiotic. Experiments to investigate the stoichiometry of the GenK reaction revealed that one equivalent each of 5′-deoxyadenosine and S-adenosyl-homocysteine are produced for each methylation reaction catalyzed by GenK. Furthermore, isotope-labeling experiments demonstrate that the S-methyl group from SAM is transferred to Cbl and the aminoglycoside product during the course of the reaction. Based on these results, one mechanistic possibility for the GenK reaction can be ruled out and further questions regarding the mechanisms of Cbl-dependent radical SAM methyltransferases, in general, are discussed.

A 4-oxalocrotonate tautomerase (4-OT) trimer has been isolated from Burkholderia lata and a kinetic, mechanistic, and structural analysis has been performed. The enzyme is the third described oligomer state for 4-OT along with a homo-and heterohexamer. The 4-OT trimer is part of a small subset of sequences (133 sequences) within the 4-OT subgroup of the tautomerase superfamily (TSF). The TSF has two distinct features: members are composed of a single β-α-β unit (homo-and heterohexamer) or two consecutively joined β-α-β units (trimer) and generally have a catalytic amino-terminal proline. The enzyme, designated fused 4-OT, functions as a 4-OT where the active site groups (Pro-1, Arg-39, Arg-76, Phe-115, Arg-127) mirror those in the canonical 4-OT from Pseudomonas putida mt-2. Inactivation by 2-oxo-3-pentynoate suggests that Pro-1 of fused 4-OT has a low pK a enabling the prolyl nitrogen to function as a general base. A remarkable feature of the fused 4-OT is the absence of P3 rotational symmetry in the structure (1.5 Å resolution). The asymmetric arrangement of the trimer is not due to the fusion of the two β-α-β building blocks because an engineered "unfused" variant that breaks the covalent bond between the two units (to generate a heterohexamer) assumes the same asymmetric oligomerization state. It

C3′-deoxygenation of aminoglycosides results in their decreased susceptibility to phosphorylation thereby increasing their efficacy as antibiotics. However, the biosynthetic mechanism of C3′-deoxygenation is unknown. To address this question, aprD4 and aprD3 genes from the apramycin gene cluster in Streptomyces tenebrarius were expressed in E. coli and the resulting gene products were characterized in vitro. AprD4 is shown to be a radical SAM enzyme catalyzing homolysis of SAM to 5′-deoxyadenosine (5′-dAdo) in the presence of paromamine. [4′-2H]-Paromamine was prepared and used to demonstrate that its C4′-H is transferred to 5′-dAdo by AprD4, during which the substrate is dehydrated to a product consistent with 4′-oxolividamine. In contrast, paromamine is reduced to a deoxy product when incubated with AprD4/AprD3/ NADPH. These results indicate that AprD4 is the first radical SAM diol-dehydratase and along with AprD3 is responsible for 3′-deoxygenation in the biosynthesis of aminoglycosides.

A Pseudomonas sp. UW4_01740 (designated Ps01740) protein of unknown function was identified as similar to 4-oxalocrotonate tautomerase (4-OT)-like and cis-3-chloroacrylic acid dehalogenase (cis-CaaD)-like subgroups of the tautomerase superfamily (TSF). Ps01740 lacks only Tyr-103 of the amino acids critical for cis-CaaD activity (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, Glu-114). As Ps01740 may represent an important variant of these enzymes, its kinetic and structural properties have been determined. Ps01740 shows tautomerase activity with phenylenolpyruvate, but lacks native 4-OT activity and dehalogenase activity with the isomers of 3-chloroacrylic acid. Ps01740 shows mostly low-level hydratase activity at pH 7.0, converting 2-oxo-3-pentynoate to acetopyruvate, consistent with cis-CaaD-like behavior. At pH 9.0, this compound results primarily in covalent modification of Pro-1, which is consistent with 4-OT-like behavior. These observations could reflect a pKa for Pro-1 that is closer to that of cis-CaaD (~9.2) than to 4-OT (~6.4. A structure of the native enzyme, at 2.6 Å resolution, highlights differences at the active site from those of 4-OT and cis-CaaD that add to our understanding of how contemporary TSF reactions and mechanisms may have diverged from a common 4-OT-like ancestor.