Products Cited in Publications Worldwide A DEIODINASE BRANCHES OUT: REDUCTION OF NITROAROMATICS TO FACILITATE BIOREMEDIATION, AND DEBROMINATION IN DROSOPHILA TO CONTROL MALE FERTILITY

A DEIODINASE BRANCHES OUT: REDUCTION OF NITROAROMATICS TO FACILITATE BIOREMEDIATION, AND DEBROMINATION IN DROSOPHILA TO CONTROL MALE FERTILITY

Qi Su

Abstract

Iodotyrosine deiodinases (IYDs) are versatile flavoenzymes. IYD was first discovered in humans as a deiodinase involved in iodide salvage for thyroid hormone biosynthesis. Mechanistic study suggested that IYD catalyzed deiodination of mono- and di-iodotyrosine using a series of single electron transfer. In the first part of this thesis, IYDs from a range of organisms were evaluated for their ability to catalyze nitroreduction by cofactor engineering. The cofactor FMN was replaced by 5-deaza FMN to suppress the sequential one-electron transfer for deiodination by 10000-fold and to promote a non-natural nitroreduction activity with a turnover frequency of 1.4 min-1. The engineered IYD∙5dFMN reduced nitroaromatics to aromatic amines catalytically using NaBH4 as a reducing source. The full 6-electron reduction of nitroaromatics to amines is rarely possible by other nitroreductases (NRs). In the second part of this thesis, the biological function of IYD was investigated in a model invertebrate, Drosophila melanogaster, which does not have a thyroid nor known to require iodide. A knockout or inactivated mutation of the cdt gene (encoding IYD) in male Drosophila using CRISPRCas9 gene editing suppressed fecundity by more than 90% over 6 days. The role of IYD in Drosophila spermatogenesis was studied using a combination of LC-MS based metabolite profiling, immunofluorescent imaging and genetic rescue. Drosophila IYD was previously known to promote debromination and dechlorination as well as deiodination. Accumulation of mono- and di-bromotyrosine at nanomolar concentrations was detected from either IYD suppression or the presence of excess bromotyrosine in diet. This accumulation disrupted over 80% of the assembly of actin cones in Drosophila testes and led to defects in spermatids individualization. Overexpression of transgenic IYD lowered the concentration of bromotyrosine in testes, restored normal spermatids individualization and rescued Drosophila fertility. Taken together, IYD is proposed to affect Drosophila fertility by regulating bromotyrosine concentration and normal spermatids individualization.  The many faces of IYD functions beyond deiodination and iodide recycling should facilitate the application of IYD in bioremediation and studies of coevolution of halogenated compounds and dehalogenase in biology.

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2-Amino-4-(trifluoromethyl)phenol

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