Investigating breadth of the rid protein superfamily
Hanson, Kelsey Marie Hodge
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The Rid (YjgF/YER057c/UK114) protein superfamily is ubiquitous and has been the subject of numerous structure-function efforts. The role of this superfamily remained unknown until work in bacteria elucidated a biochemical role for the RidA protein in endogenous stress response. Recent bioinformatics and phylogenetic analyses indicated the superfamily is composed of eight subgroups (RidA and Rid1-7). The archetypal member of the RidA subfamily, RidA from Salmonella enterica, is a reactive enamine/imine deaminase that mediates metabolic stress from the reactive metabolite 2-aminoacrylate (2AA). The majority of the structural, biochemical, and genetic data were obtained with members of the RidA subfamily. Our understanding is in the early stages for the remaining subfamilies. The importance of RidA is reflected in the mutant phenotypes of bacteria, plants, and yeast that lack a relevant RidA homolog. Generation of 2AA, as well as the negative downstream consequences, is linked to the essential cofactor pyridoxal 5’-phosphate (PLP). Previous work has shown that 2AA deaminase activity is conserved in RidA homologs from all domains of life. This work addressed the breadth of the Rid superfamily, including biochemical characterization of Rid proteins from subfamilies Rid1, 2, and 3, that showed Rid proteins deaminate multiple substrates in addition to 2AA in vitro. The substrate specificities of Rid2 and Rid3 protein were distinct from those of RidA and Rid1 subfamilies and these differences were explored by mutational analysis and biochemical analysis of variant proteins. A link was uncovered between Rid proteins and flavin adenine dinucleotide (FAD)-dependent enzymes. D-arginine dehydrogenase was identified as the third mechanism for bacterial generation of Rid substrates and this interaction was reconstituted in vitro. A heterologous complementation experiment identified a protein from the methanogenic archaeon Methanococcus maripaludis that uncovered an indirect mechanism for 2AA quenching in bacteria. Together, the ideas in this dissertation support the idea that Rid proteins facilitate deamination of reactive catalytic intermediates in multiple areas of metabolism.