A distinguishing trait of heme enzymes is that a high-valent iron-oxo species is a common oxidant for mediating a remarkable array of oxidation reactions. However, one conundrum is that each enzyme, in general, promotes only a specific type of reaction. How the reaction type is determined after the formation of the key oxidant remains an open question whose answers have implications for our fundamental understanding of enzyme catalysis as well as de novo enzyme design, protein engineering, and rationally designed inorganic catalysts. Because tyrosine is an important building block of natural products, this application focuses on the 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 the more common hydroxylation reaction. We found that SfmD is a new member of the tryptophan dioxygenase superfamily that promotes regioselective monooxygenation of a methylated tyrosine substrate. The peroxidase LmbB2 performs a peroxygenase-type reaction with an axial histidine ligand rather than cysteine. These enzymes catalyze tyrosine-based oxidation reactions and are related to antimicrobial drug development. Given the similarities of the heme-based oxidant and the structure of the substrates, the inevitable question arises 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 substrate positioning relative to the iron-bound oxidant and the capture of catalytic intermediates. We have recently identified that this protein is a novel heme-based oxygenase.
Aim #3 is focused on studying the peroxygenase- type 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 hypotheses. An 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, and reveal underlying mechanisms to further aid rational drug design and discovery processes.
The proposed studies are relevant to human health because the enzymes under investigation are crucial to the synthesis of natural compounds that are antibiotic, antitumor agent, or essential for pathogens such as Mycobacterium tuberculosis, i.e., lincomycin, saframycin A, and mycocyclosin. The insight gained from these studies will aid the design of new therapeutic compounds that could lead to better treatments for infections from bacterial pathogens and cancer.
Showing the most recent 10 out of 22 publications
