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 and protein engineering. Because tyrosine is an important building block of natural products, this application 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 a 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 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, the inevitable question arises regarding the governing factors that determine the catalytic activity of these enzymes.
In Aim #1, we will determine 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 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 is focused 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 hypotheses. 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.

Public Health Relevance

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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function A Study Section (MSFA)
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Barski, Oleg
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University of Texas Health Science Center San Antonio
Schools of Arts and Sciences
San Antonio
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Li, Jiasong; Griffith, Wendell P; Davis, Ian et al. (2018) Cleavage of a carbon-fluorine bond by an engineered cysteine dioxygenase. Nat Chem Biol 14:853-860
Shin, Inchul; Ambler, Brett R; Wherritt, Daniel et al. (2018) Stepwise O-Atom Transfer in Heme-Based Tryptophan Dioxygenase: Role of Substrate Ammonium in Epoxide Ring Opening. J Am Chem Soc 140:4372-4379
Davis, Ian; Yang, Yu; Wherritt, Daniel et al. (2018) Reassignment of the human aldehyde dehydrogenase ALDH8A1 (ALDH12) to the kynurenine pathway in tryptophan catabolism. J Biol Chem 293:9594-9603
Wang, Yifan; Griffith, Wendell P; Li, Jiasong et al. (2018) Cofactor Biogenesis in Cysteamine Dioxygenase: C-F Bond Cleavage with Genetically Incorporated Unnatural Tyrosine. Angew Chem Int Ed Engl 57:8149-8153
Yang, Yu; Liu, Fange; Liu, Aimin (2018) Adapting to oxygen: 3-Hydroxyanthrinilate 3,4-dioxygenase employs loop dynamics to accommodate two substrates with disparate polarities. J Biol Chem 293:10415-10424
Davis, Ian; Koto, Teruaki; Liu, Aimin (2018) Radical Trapping Study of the Relaxation of bis-Fe(IV) MauG. React Oxyg Species (Apex) 5:46-55
Davis, Ian; Koto, Teruaki; Terrell, James R et al. (2018) High-Frequency/High-Field Electron Paramagnetic Resonance and Theoretical Studies of Tryptophan-Based Radicals. J Phys Chem A 122:3170-3176
Krishnan, V Mahesh; Davis, Ian; Baker, Tessa M et al. (2018) Backbone Dehydrogenation in Pyrrole-Based Pincer Ligands. Inorg Chem 57:9544-9553
Dornevil, Kednerlin; Davis, Ian; Fielding, Andrew J et al. (2017) Cross-linking of dicyclotyrosine by the cytochrome P450 enzyme CYP121 from Mycobacterium tuberculosis proceeds through a catalytic shunt pathway. J Biol Chem 292:13645-13657
Ferreira, Patrick; Shin, Inchul; Sosova, Iveta et al. (2017) Hypertryptophanemia due to tryptophan 2,3-dioxygenase deficiency. Mol Genet Metab 120:317-324

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