Project 2

One, Two and Three Rieske Routes for Catalyzing Site-Specific Oxygenations

Nature has perfected an arsenal of strategies to functionalize inert C-H bonds. This chemistry is fundamental to a range of metabolic pathways and has evolved to be selective, controlled, and to showcase precision that is often difficult to achieve synthetically. Nature’s strategies often require the use of a metalloenzyme to exploit molecular oxygen (O2) for formation of a potent oxidant1,2. Once formed, these oxidants can be used to abstract a hydrogen atom from an unactivated substrate and initiate the incorporation of one (monooxygenases) or two (dioxygenases) oxygen atoms from O2 into a product1,2. One of Nature’s most ubiquitous and underexplored strategies for facilitating mono- and di-oxygenation reactions is observed in the Rieske oxygenase enzyme class. These enzymes couple a [2Fe-2S] cluster with a non-heme iron site (see below) to perform a mechanistically diverse set of powerful, efficient, and site-specific transformations at traditionally inert centers.

These enzymes function as monooxygenases, dioxygenases, and have even been demonstrated to facilitate multiple single monooxygenation reactions (see above)3-6. In addition, these enzymes have been implicated in C-C bond formation7, dealkylation8, and several other types of challenging reactions. The chemistry performed by Rieske oxygenases plays prominent roles in biosynthetic pathways that produce medically and commercially valuable compounds, as well as in degradative pathways that remove pollutants from the environment. Therefore, these enzymes represent a valuable source of enzymatic strategies to industrially produce pharmaceuticals and commodity chemicals, or facilitate bioremediation efforts. However, the practical applicability of these enzymes is limited by the lack of information available about the structure–function relationships in this class of enzymes.

One of the key questions our lab is interested in answering is:

How does one set of metallocenters facilitate such a diverse range of chemistry?

To answer this question, we are using enzymology, spectroscopy, reduction potential measurements, and structural biology. Our goal is to unravel the many mysteries about how Rieske oxygenases exquisitely control activated oxygen species to facilitate site-specific reactions with diverse outcomes. We hypothesize the answers lie in understanding how regions outside of the active site facilitate catalysis, how the metallocenters are tuned to form the needed oxidant, and how different regions of the protein communicate. Our goal is to identify trends that will lend predictive power to repurposing and redesigning these enzymes for biotechnological purposes.

 

 

References:

  1. Ray, K., Pfaff, F. F., Wang, B. & Nam, W. Status of reactive non-heme metal-oxygen intermediates in chemical and enzymatic reactions. J Am Chem Soc 136, 13942-13958, (2014).
  2. Li, B. & Bridwell-Rabb, J. Aerobic Enzymes and Their Radical SAM Enzyme Counterparts in Tetrapyrrole Pathways. Biochemistry 58, 85-93, (2019).
  3. Junker, F., Kiewitz, R. & Cook, A. M. Characterization of the p-toluenesulfonate operon tsaMBCD and tsaR in Comamonas testosteroni T-2. J Bacteriol 179, 919-927 (1997).
  4. Hibino, T., Waditee, R., Araki, E., Ishikawa, H., Aoki, K., Tanaka, Y. & Takabe, T. Functional characterization of choline monooxygenase, an enzyme for betaine synthesis in plants. J Biol Chem 277, 41352-41360, (2002).
  5. Porra, R. J., Schafer, W., Cmiel, E., Katheder, I. & Scheer, H. Derivation of the formyl-group oxygen of chlorophyll b from molecular oxygen in greening leaves of a higher plant (Zea mays). FEBS Lett 323, 31-34 (1993).
  6. Lee, J., Simurdiak, M. & Zhao, H. Reconstitution and characterization of aminopyrrolnitrin oxygenase, a Rieske N-oxygenase that catalyzes unusual arylamine oxidation. J Biol Chem 280, 36719-36727, (2005).
  7. Higgins, T. P., Demarco, P. & Murrell, J. C. Purification and molecular characterization of the electron transfer protein of methanesulfonic acid monooxygenase. J. Bacteriol. 179, 1974-1979 (1997).
  8. Withall, D. M., Haynes, S. W. & Challis, G. L. Stereochemistry and mechanism of undecylprodigiosin oxidative carbocyclization to streptorubin B by the Rieske oxygenase RedG. J. Am. Chem. Soc. 137, 7889-7897 (2015)