Protein-derived cofactor in bifunctional enzyme KatG from Mycobacterium tuberculosis Project Description The catalase-peroxidase enzyme KatG is a heme-dependent protein critical for the virulence of Mycobacterium tuberculosis (Mtb). Its catalase function is essential for the pathogen to mitigate oxidative stress from hydrogen peroxide in infected host cells. The catalase activity of KatG is distinct from other catalases due to the presence of a protein-derived cofactor, methionine-tyrosine-tryptophan (MYW) covalent triad, which is an auto-catalytical post-translationally modification. Because of the MYW cofactor, the catalase activity is more efficient by four orders of magnitude compared to its peroxidase activity. However, KatG, as the sole catalase active protein in Mtb and a key defense against oxidative damage by human host defenses, has not been appreciated. This is primarily because the anti-tuberculosis prodrug isoniazid has been the primary focus of Mtb treatment. Isoniazid takes advantage of the peroxidase activity for its activation. Here, we consider that the catalase function of KatG may be targeted to provide a novel two-pronged assault on Mtb. The inhibition of catalase activity can directly affect the virulence of Mtb, while also potentiating the peroxidase activity to boost isoniazid activation by KatG. With this motivation, we have invested heavily to further our understanding of the MYW cofactor. Our preliminary studies found that the protein-derived cofactor in KatG is naturally present in two forms. It is mainly in an MYW- OOH form at ambient temperature and mild growth conditions. While at body temperature, it is primarily in the catalase active MYW form. The MYW-OOH-containing KatG has little catalase activity but can autocatalytically convert to the MYW-containing KatG when it encounters concentrated hydrogen peroxide, instinctually obtaining catalase activity. Following these findings, we will further investigate the chemical nature of the protein-derived cofactor in KatG using various experimental and computational approaches. Specifically, we will 1) examine the chemical nature of the cofactor in the two forms and the conversion pathway from the catalase-inactive state to its active form to inform why and how a hydroperoxyl group can be added/removed to an indole-nitrogen, an unprecedented chemical process in a biological system, and what may be the chemical consequence of the catalytic functions of KatG with an additional oxygenation modification of the cofactor, 2) dissect the role of the crosslinked amino acid components of the cofactor, elucidate electronic and charge contribution in catalysis through strategical alterations by using site-specific genetic substitution of non-canonical amino acids, and evaluate how the strategically altered cofactors may distribute the oxidizing power distinctly and how they would affect isoniazid activation, and 3) investigate the role of the heme environment cofactor biosynthesis a...