The minimal structure for iodotyrosine deiodinase function is defined by an outlier protein from the thermophilic bacterium Thermotoga neapolitana

Abstract

The nitroreductase superfamily of enzymes encompasses many flavin mononucleotide (FMN)-dependent catalysts promoting a wide range of reactions. All share a common core consisting of an FMN-binding domain, and individual subgroups additionally contain one to three sequence extensions radiating from defined positions within this core to support their unique catalytic properties. To identify the minimum structure required for activity in the iodotyrosine deiodinase subgroup of this superfamily, attention was directed to a representative from the thermophilic organism Thermotoga neapolitana (TnIYD). This representative was selected based on its status as an outlier of the subgroup arising from its deficiency in certain standard motifs evident in all homologues from mesophiles. We found that TnIYD lacked a typical N-terminal sequence and one of its two characteristic sequence extensions, neither of which was found to be necessary for activity. We also show that TnIYD efficiently promotes dehalogenation of iodo-, bromo-, and chlorotyrosine, analogous to related deiodinases (IYDs) from humans and other mesophiles. In addition, 2-iodophenol is a weak substrate for TnIYD as it was for all other IYDs characterized to date. Consistent with enzymes from thermophilic organisms, we observed that TnIYD adopts a compact fold and low surface area compared with IYDs from mesophilic organisms. The insights gained from our investigations on TnIYD demonstrate the advantages of focusing on sequences that diverge from conventional standards to uncover the minimum essentials for activity. We conclude that TnIYD now represents a superior starting structure for future efforts to engineer a stable dehalogenase targeting halophenols of environmental concern.

Substrate Electronics Dominate the Rate of Reductive Dehalogenation Promoted by the Flavin-Dependent Iodotyrosine Deiodinase

“…Protonation of the aromatic ring of I-Tyr is an obvious process that can limit turnover since the loss of aromaticity is highly endergonic even for the electron-rich systems such as a phenol. Since no proton donor has yet been identified in the active sites of various IYDs, ,,, site-directed mutagenesis is ineffective for examining such protonation. However, the contribution of proton transfer to the overall rate of turnover can be measured by solvent kinetic isotope effects irrespective of the specific donor.…”

Sequence Conservation Does Not Always Signify a Functional Imperative as Observed in the Nitroreductase Superfamily

“…However, this leads to a broader question about the extent to which the functional importance of residues may correlate to their conservation in certain subfamilies or throughout an entire superfamily of proteins. Already, our laboratory has demonstrated that an active site loop in IYD was not necessary for dehalogenation despite its predominance in the IYD subfamily . We have now generalized this analysis in the current report to evaluate if the dominance of a particular residue common to most of the NTR superfamily is necessarily linked to a functional imperative.…”

“…Bulk solvent could suppress such an effect in many enzymes but is excluded in IYD when the substrate is bound and the active site lid is closed. 11,19,27…”

“…Thus, an acidic proton from a neutral side chain would become available to coordinate FMN and stabilize its reduced forms. Bulk solvent could suppress such an effect in many enzymes but is excluded in IYD when the substrate is bound and the active site lid is closed. ,, …”

“…Already, our laboratory has demonstrated that an active site loop in IYD was not necessary for dehalogenation despite its predominance in the IYD subfamily. 19 We have now generalized this analysis in the current report to evaluate if the dominance of a particular residue common to most of the NTR superfamily is necessarily linked to a functional imperative. An Arg or Lys is positioned adjacent to the N1 and C2 carbonyl of FMN throughout the superfamily and selected for analysis, as described below.…”