A SEARCH FOR THE RATE DETERMINING STEP OF IODOTYROSINE DEIODINASE CATALYSIS AND MINIMAL REQUIREMENT FOR ENZYME CATALYZED DEIODINATION

Iodotyrosine deiodinase (IYD) represents the only reductive dehalogenase in mammals that requires flavin for catalysis. This enzyme was first identified in the 1950s, but its catalytic mechanism still remains poorly understood. Our proposed mechanism involves protonation of the halotyrosine from its enolate to keto form. To support this possibility, a series of pyridone analogues were designed and synthesized to mimic the nonaromatic keto tautomer of iodotyrosine (I-Tyr). These compounds bind more tightly to the reduced form (FMNhq) of IYD than to its oxidized form (FMNox) by as much as 3,200 to 45,000-fold. Such difference can be explained by a change in IYD conformation upon the reduction of its FMN cofactor. In contrast, the inert substrate analog fluorotyrosine exhibits only a 5-fold difference in affinity for the oxidized and reduced forms of IYD. Deiodination of I-Tyr by IYD is not a fast process, and the rate determining features for this reaction were not previously identified. To examine the potential for substrate protonation to be rate determining, the solvent deuterium isotope effect was measured, and kcat(H2O)/kcat(D2O) was found to be 1.23±0.15 under steady state conditions, indicating that protonation of the substrate is not a major contributor to the rate limiting step. Similarly, reconstitution of IYD with FMN analogs with redox potentials ranged from -128 mV to -260 mV affected kcat by no more than 3-fold. These data suggests that electron transfer is also not the major contributor to the rate determining step. The possibility that closure of an active site lid could be rate limiting was also tested. However, conjugation of IYD to APM, a fluorescent probe for studying conformational changes in enzymes, did not allow for monitoring the rate of IYD active site lid closure. Reconstitution of Thermotoga neapolitana IYD (TnIYD) with 2’-deoxyFMN allowed the enhancement of hydride transfer of this modified enzyme compared to wt (wild type) enzyme. 23% more of 2’-deoxyFMN TnIYD was reduced compared to the wt enzyme using the same amount of NaBH4. Moreover, the modified enzyme, unlike wt TnIYD, was fully oxidized by NO2-Tyr during a single-turnover experiment. At the same time, the single electron transfer ability of 2’-deoxyFMN TnIYD was preserved, and it was still able to deiodinate I-Tyr with a similar kcat compared to wt TnIYD. At the same time, the KM (I-Tyr) value for 2’-deoxyFMN TnIYD decreased by 1,200-fold compared to the wt enzyme and suggested that 2’-OH also plays an important role in substrate binding. IYD belongs to the nitroreductase (NR) superfamily and shares structural similarities with FMN-containing NRs, but it is unable to catalyze nitroreduction. NRs are known to have a very broad substrate specificity, so a dehalogenase created from such a NR can find an application in bioremediation of halophenols that are used as pesticides and continue to accumulate in the environment. The possibility of turning an NR into a dehalogenase by stabilizing its FMNsq during catalysis was tested. Stabilization of FMNsq was proposed to be important for IYD catalyzed dehalogenation. Two different strategies were used to stabilize FMNsq for NRs. Firstly, an E165S mutation for NfsB was proposed to install a strong hydrogen bond from a serine residue to N5 of FMN. Second, reconstitution of NfsB with 6-carboxyFMN was proposed, since the carboxy group could form an internal hydrogen bond to N5 of FMN for stabilizing FMNsq. Unfortunately, no deiodination was detected for E165S NfsB, and the synthesis of 6-carboxyFMN is still ongoin