Date of Award
Master of Science
Dr. Frederick Stull
Dr. David Huffman
Dr. Todd Barkman
Enzymology, oxidative stress resistance, RcIA, HOSCN, bacteria
Masters Thesis-Open Access
Reactive chlorine species (RCS) - hypochlorous acid (HOCl), hypothiocyanous acid (HOSCN), and chloramines - are powerful antimicrobial oxidants generated by the innate immune system. Bacteria developed defense mechanisms against RCS, which are highly toxic for invading pathogens. One such defense system involves RclA, an enzyme from the flavin disulfide reductase family, which is massively upregulated upon exposure of Escherichia coli to RCS. Recent research suggests that RclA functions as a Cu(II) reductase to provide resistance against RCS. However, the exact mechanism used by RclA was subject to debate. In this study, we demonstrate that RclA is unlikely to function as a Cu(II) reductase. Instead, we show that RclA provides resistance against RCS by directly detoxifying HOSCN, reducing it to non-toxic thiocyanate (SCN-) using NAD(P)H via a ping-pong kinetic mechanism. RclA catalyzes the reduction of HOSCN with kcat of 182 s-1, and the specificity constant (kcat/Km) of 9.12 x 107 M-1 s-1, indicating that RclA is a potent and highly specific HOSCN reductase. HOSCN is a primarily sulfhydryl-reactive oxidant, and RclA has two conserved cysteine residues at its active site. Thus, we hypothesize that RclA detoxifies HOSCN before it can target other thiols in the cell, and the enzyme’s active site cysteines are critical for its HOSCN reductase activity. Our study provides a rationale for how bacteria can cope with RCS produced by the immune system. Targeting RclA in bacteria found in oral cavity or lung airway fluids may make pathogenic bacteria more susceptible to the immune response, thus allowing the creation of new strategies for controlling pathogenic infections.
Chapman, Irina, "The Flavoprotein RCLA is a Hypothiocyanous Acid Reductase" (2021). Masters Theses. 5234.