Conversion of a Dehalogenase into a Nitroreductase by Swapping its Flavin Cofactor with a 5‐Deazaflavin Analogue

Abstract

Natural and engineered nitroreductases have rarely supported full reduction of nitroaromatics to their amine products, and more typically, transformations are limited to formation of the hydroxylamine intermediates. Efficient use of these enzymes also requires a regenerating system for NAD(P)H to avoid the costs associated with this natural reductant. Iodotyrosine deiodinase is a member of the same structural superfamily as many nitroreductases but does not directly consume reducing equivalents from NAD(P)H, nor demonstrate nitroreductase activity. However, exchange of its flavin cofactor with a 5-deazaflavin analogue dramatically suppresses its native deiodinase activity and leads to significant nitroreductase activity that supports full reduction to an amine product in the presence of the convenient and inexpensive NaBH .

The distribution and mechanism of iodotyrosine deiodinase defied expectations

“…A similar partial oxidation of FMN hq to FMN sq was evident after addition of O 2 N-Tyr to human IYD but no consumption of O 2 N-Tyr was observed in this example [54]. The lack of nitro group reduction was not particularly surprising since nitroreductases seem to promote reduction through hydride transfer [55, 56] whereas IYD promotes reduction through single electron transfers [31, 36, 54]. The ability of the flavin destructase BluB to activate molecular oxygen suggests its ability to promote single electron transfer from its flavin but this enzyme neither promotes a detectable level of I 2 -Tyr deiodination nor does IYD promote a detectable level of flavin destructase activity [28].…”

“…Some consumption of O 2 N-Tyr was noted in this case but its origins remains unknown [36]. A similar partial oxidation of FMN hq to FMN sq was evident after addition of O 2 N-Tyr to human IYD but no consumption of O 2 N-Tyr was observed in this example [54]. The lack of nitro group reduction was not particularly surprising since nitroreductases seem to promote reduction through hydride transfer [55, 56] whereas IYD promotes reduction through single electron transfers [31, 36, 54].…”

“…Loss of this interaction may suppress accumulation and detection of FMN sq but does not preclude its transient formation during catalysis. Single electron transfer is still the favored mechanism of dehalogenation since substitution of FMN with 5-deazaFMN decreases the k cat of native IYD by greater than 10 5 -fold [54]. This deazaFMN is well known to support the two electron chemistry of FMN but incapable of participating in its one electron chemistry [85, 86].…”

“…Thus, a very minimal change in sequence has the potential to create a significant change in activity and suggests that evolution of these enzyme families required only a slight variation to start them on their unique paths of development. Consistent with the dichotomy of dehalogenase versus nitroreductase and one versus two electron transfer, reconstitution of IYD with 5-deazaFMN also acts as a nitroreductase and promotes full reduction of O 2 N-Tyr to H 2 N-Tyr [54]. …”

Broadening the Scope of the Flavin‐Tag Method by Improving Flavin Incorporation and Incorporating Flavin Analogs

“…Roseoflavin was ordered from Toronto Research Chemicals. 5‐Deazariboflavin was chemically synthesized using a previously published protocol (Figure S14) [22] . All other chemicals were ordered from Sigma‐Aldrich.…”