Heme-dependent chemistry in tyrosine oxidation: Diversity Supplement on 5R01GM108988-08 for supporting Samuel Montoya

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Abstract of the Parent Research Project One of the hallmarks of heme iron-dependent enzymes is the wide array of oxidation reactions that it can catalyze with its high-valent iron-oxo species. However, one conundrum is that each protein, in general, promotes only a specific type of reaction. How the reaction type is determined after the formation of the critical oxidant remains an open question whose answers have implications for the fundamental understanding of enzyme catalysis as well as de novo enzyme design and protein engineering. Because tyrosine is an essential building block of natural products, this project focuses on a mechanistic characterization of three heme-dependent tyrosine-oxidizing enzymes. Each of these enzymes employs a mononuclear heme cofactor to oxidize its tyrosine-based substrate. Intriguingly, a cytochrome P450 protein, CYP121 from Mycobacterium tuberculosis, catalyzes an unusual oxidative carbon-carbon cross- coupling reaction instead of a more common hydroxylation reaction. We found that SfmD is a new member of the tryptophan dioxygenase superfamily that promotes a regioselective monooxygenation on a methylated tyrosine substrate. The peroxidase LmbB2 performs a peroxygenase-type of reaction with an axial ligand of histidine instead of cysteine. These enzymes not only catalyze tyrosine-based oxidation reactions, but they are also related to antimicrobial drug development. Given the similarities of the heme- based oxidant and the structure of the substrates, an inevitable question arises and will be answered by this study regarding the governing factors that determine the catalytic activity of these enzymes. In Aim #1, we will identify the mechanistic and structural characteristics of CYP121. Using a battery of spectroscopic and structural approaches coupled with synthetic probes, we will unveil a novel carbon- carbon coupling mechanism mediated by the P450 enzyme. In Aim #2, we will characterize the structure and mechanism of SfmD with an emphasis on how the substrate is positioned to the iron-bound oxidant and the capture of catalytic intermediates. We have already identified that this protein is a novel heme- based oxygenase. Aim #3 will focus on studying the peroxidase reaction catalyzed by LmbB2 that is responsible for L-3,4-dihydroxyphenylalanine (L-DOPA) formation through L-tyrosine hydroxylation. We will utilize small-molecule probes to interrogate mechanistic pathways. The in-depth analysis of these three related catalytic systems will test our hypothesis regarding how the heme-bound oxidant is generated and directed to the aromatic substrates, unravel the structure-function relationships of the heme enzymes of seemingly unrelated superfamilies at a higher level, and develop underlying mechanisms further aiding rational drug design and discovery processes.