Bollinger, J. Martin Jr. - The Pennsylvania State University

研究领域

Mechanisms of Metalloenzymes and Metallocofactor Assembly Our research concerns a fascinating group of proteins that contain complex clusters of metal ions and inorganic ligands at their active sites. These proteins play essential roles in such crucial biochemical processes as nitrogen fixation, photosynthesis, oxidative phosphorylation and ribonucleotide reduction. We aim to understand the relationship between the structures and catalytic mechanisms of enzymes that employ such clusters, and to elucidate the biochemical mechanisms by which the proteins acquire their clusters. Currently, the majority of our efforts are in two areas. In the first, we are defining structure-reactivity relationships among members of a large class of enzymes that use carboxylate-bridged dinuclear iron clusters (see Figure 1, top) to activate molecular oxygen for diverse oxidation reactions. Several of these reactions, which range from hydrocarbon hydroxylation (e.g. methane monooxygenase, MMO) to fatty acid desaturation (e.g. stearoyl acyl carrier protein delta-9 desaturase, delta-9-D) to generation of a stable tyrosyl radical (in subunit R2 of ribonucleotide reductase, R2-RNR), are considered to be difficult because no counterparts exist in the repertoire of the synthetic chemist, in spite of the significant effort that has been directed toward their development. Our interest stems primarily from the recent discovery that members of this class, while catalyzing quite distinct oxidation reactions, have very similar structures. For example, the di-iron clusters of R2-RNR and MMO (Figure 1, top) have an identical ligand set (with the exception of a single conservative Glu to Asp substitution), and the protein folds defining their cluster sites (Figure 1, botttom) are quite similar. Thus, the central question which drives our research on these proteins can be formulated: how do the various proteins "tune" a single catalytic motif for this remarkably diverse set of oxidation reactions? Our approach to answering this question is 1) to compare the reaction mechanisms that have been defined for members of the class, 2) to combine the mechanistic information with the available structural data in order to formulate detailed hypotheses as to the elements of each protein and di-iron cluster that distinguish its reaction mechanism and outcome, 3) to construct, on the basis of these hypotheses, mutant proteins designed to have altered mechanisms and outcomes, and 4) to define the altered mechanisms and outcomes of these engineered proteins in order to test the design hypotheses. To achieve the latter, we and our collaborators bring to bear a dazzling array of biophysical tools, including state-of-the-art rapid kinetic methods (stopped-flow, chemical quench, and freeze quench methods) and spectrosopic methods (EPR, ENDOR, EXAFS, resonance Raman, CD/MCD, x-ray crystallography). The "holy grail" of the project is to learn how to rationally alter one protein of the class to have a mechanism and outcome mimicking that of another member. Such an understanding of the molecular logic underlying "tuning" of di-iron cluster reactivity would aid significantly in current efforts to develop biomimetic oxidation catalysts based on this unit.

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Rajakovich, L. J.; Zhang, B.; McBride, M. J., Boal, A. K.; Krebs, C.; Bollinger, J. M., Jr. “Emerging Structural and Functional Diversity in Proteins with Dioxygen-Reactive Dinuclear Transition Metal Cofactors” in “Comprehensive Natural Products III: Chemistry and Biology” edited by Liu, H.-w. and Begley, T. P., Elsevier, Amsterdam, 2020. [link to publisher’s website] Cutsail, G. E., III; Blaesi, E. J.; Pollock, C. J.; Bollinger, J. M., Jr.; Krebs, C.; DeBeer, S. “High-Resolution Iron X-ray Absorption Spectroscopic and Computational Studies of Non-Heme Diiron Peroxo Intermediates” J. Inorg. Biochem. 2020, 203, 110877. [link to publisher’s website] Zhou, S.; Pan, J.; Davis, K. M.; Schaperdoth, I.; Wang, B.; Boal,, A. K.; Krebs, C.; Bollinger, J. M., Jr. “Steric Enforcement of cis-Epoxide Formation in the Radical C–O-Coupling Reaction by which (S)-2-Hydroxypropylphosphonate Epoxidase (HppE) Produces Fosfomycin” J. Am. Chem. Soc. 2019, 141, 51, 20397-20406. [link to publisher’s website] Davis, K. M.; Altmyer, M.; Martinie, R. J.; Schaperdoth, I.; Krebs, C.; Bollinger, J. M., Jr.; Boal, A. K. “Structure of a Ferryl Mimic in the Archetypal Iron(II)- and 2-(Oxo)-Glutarate-Dependent Dioxygenase, TauD” Biochemistry 2019, 58, 4218-4223. [link to publisher’s website] Pan, J.; Wenger, E. S.; Matthews, M. L.; Pollock, C. J.; Bhardwaj, M.; Kim, A. J., Allen, B. D.; Grossman, R. B.; Krebs, C.; Bollinger, J. M., Jr. “Evidence for Modulation of Oxygen-Rebound Rate in Control of Outcome by Iron(II)- and 2-Oxoglutarate-Dependent Oxygenases” J. Am. Chem. Soc. 2019, 141, 15153-15165. [link to publisher’s website] Zhang, B.; Rajakovich, L. J.; Van Cura, D.; Blaesi, E. J.; Mitchell, A. J.; Tysoe, C. R.; Zhu, X.; Streit, B. R.; Rui, Z.; Zhang, W.; Boal, A. K.; Krebs, C.; Bollinger, J. M., Jr. “Substrate-triggered Formation of a Peroxo-Fe2(III/III) Intermediate during Fatty Acid Decarboxylation by UndA” J. Am. Chem. Soc. 2019, 141, 14510-14514. [link to publisher’s website] Chekan, J. R.; Ongpipattanakul, C.; Wright, T. R.; Zhang, B.; Bollinger, J. M., Jr.; Rajakovich, L. J.; Krebs, C.; Cicchillo, R. M.; Nair, S. K. “Molecular Basis for Enantioselective Herbicide Degradation Imparted by Aryloxyalkanoate Dioxygenases in Transgenic Plants” Proc. Natl. Acad. Sci., U.S.A., 2019, 116, 13299-13304. [link to publisher’s website] Dunham, N. P.; Del Río Pantoja, J. M.; Zhang, B.; Rajakovich, L. J.; Allen, B. D.; Krebs, C.; Boal, A. K.; Bollinger, J. M., Jr. “Hydrogen Donation but not Abstraction by a Tyrosine (Y68) During Endoperoxide Installation by Verruculogen Synthase (FtmOx1)” J. Am. Chem. Soc. 2019, 141, 9964-9979. [link to publisher’s website] Rajakovich, L. J.; Pandelia, M.-E.; Mitchell, A. J.; Chang, W.-c.; Zhang, B.; Boal, A. K.; Krebs, C.; Bollinger, J. M., Jr. “A New Microbial Pathway for Organophosphonate Degradation Catalyzed by Two Previously Misannotated Non-Heme-Iron Oxygenases” Biochemistry 2019, 58, 1627-1647. [link to publisher’s website] Dunham, N. P.; Mitchell, A. J.; Del Río Pantoja, J. M.; Krebs, C.; Bollinger, J. M., Jr.; Boal, A. K. “α-Amine Desaturation of D-Arginine by the Iron(II)- and 2-(Oxo)-Glutarate-Dependent L-Arginine 3-Hydroxylase, VioC” Biochemistry 2018, 57, 6479–6488. [link to publisher’s website] Blaesi, E. J.; Palowitch, G. M.; Hu, K.; Kim, A. J.; Rose, H. R.; Alapati, R.; Lougee, M. G.; Kim, H. J.; Taguchi, A. T.; Tan, K. O.; Laremore, T. N.; Griffin, R. G.; Krebs, C.; Matthews, M. L.; Silakov, A.; Bollinger, J. M., Jr.; Allen, B. D.; Boal, A. K. “Metal-Free Class Ie Ribonucleotide Reductase from Pathogens Initiates Catalysis with a Tyrosine-Derived Dihydroxyphenylalanine Radical” Proc. Natl. Acad. Sci., U.S.A., 2018, 115, 10022-10027. [link to publisher’s website] Martinie, R. J.; Blaesi, E. J.; Bollinger, J. M., Jr.; Krebs, C.; Finkelstein, K. D.; Pollock, C. J. “Two-Color Valence-to-Core X-ray Emission Spectroscopy Tracks Cofactor Protonation State in a Class I Ribonucleotide Reductase” Angew. Chem., Int. Ed. 2018, 57, 12754-12758. [link to publisher’s website] Dunham, N. P.; Chang, W.-c.; Mitchell, A. J.; Martinie, R. J.; Zhang, B.; Bergman, J. A.; Rajakovich, L. J.; Wang, B.; Silakov, A.; Krebs, C.; Boal, A. K.; Bollinger, J. M., Jr. “Two Distinct Mechanisms for C-C Desaturation by Iron(II)- and 2-(Oxo)glutarate-Dependent Oxygenases: Importance of alpha-Heteroatom Assistance” J. Am. Chem. Soc. 2018, 140, 7116–7126. [link to publisher’s website] Rose, H. R.; Ghosh, M. K.; Maggiolo, A. O.; Pollock, C. J.; Blaesi, E. J.; Hajj, V.; Wei, Y.; Rajakovich, L. J.; Chang, W.-c.; Han, Y.; Hajj, M.; Krebs, C.; Silakov, A.; Pandelia, M.-E.; Bollinger, J. M., Jr.; Boal, A. K. “Structural Basis for Superoxide Activation of Flavobacterium johnsoniae Class I Ribonucleotide Reductase and for Radical Initiation by Its Dimanganese Cofactor” Biochemistry 2018, 57, 1372-1383. [link to publisher’s website] Pan, J.; Bhardwaj, M.; Zhang, B.; Chang, W.-c.; Schardl, C. L.; Krebs C.; Grossman, R. B.; Bollinger, J. M., Jr. “Installation of the Ether Bridge of Lolines by the Iron- and 2-Oxoglutarate-Dependent Oxygenase, LolO: Regio- and Stereochemistry of Sequential Hydroxylation and Oxacyclization Reactions” Biochemistry 2018, 57, 2074-2083. [link to publisher’s website] Kenney, G. E.; Dassama, L. M. K.; Pandelia, M.-E.; Gizzi, A. S.; Martinie, R. J.; Gao, P.; DeHart, C. J.; Schachner, L. F.; Skinner, O. S.; Ro, S. Y.; Zhu, X.; Sadek, M.; Thomas, P. M.; Almo, S. C.; Bollinger, J. M., Jr.; Krebs, C.; Kelleher, N. L.; Rosenzweig, A. C. “The Biosynthesis of Methanobactin” Science 2018, 359, 1411-1416. [link to publisher’s website] Martinie, R. J.; Pollock, C. J.; Matthews, M. L.; Bollinger, J. M., Jr.; Krebs, C.; Silakov, A. “Vanadyl as a Stable Structural Mimic of Reactive Ferryl Intermediates in Mononuclear Nonheme-Iron Enzymes” Inorg. Chem. 2017, 56, 13382–13389. [link to publisher’s website] Mitchell, A. J.; Dunham, N. P.; Martinie, R. J.; Bergman, J. A.; Pollock, C. J.; Hu, K.; Allen, B. D.; Chang, W.-c.; Silakov, A.; Bollinger, J. M., Jr.; Krebs, C.; Boal, A. K. “Visualizing the Reaction Cycle in an Iron(II)- and 2-(Oxo)-Glutarate-Dependent Hydroxylase” J. Am. Chem. Soc. 2017, 139, 13830–13836. [link to publisher’s website] Park, K.; Li, N.; Kwak, Y.; Srnec, M.; Bell, Caleb B., III ; Liu, L. V.; Wong, S. D.; Yoda, Y.; Kitao, S.; Seto, M.; Hu, M.; Zhao, J.; Krebs, C.; Bollinger, J. M., Jr.; Solomon, E. I. “Peroxide Activation for Electrophilic Reactivity by the Binuclear Non-heme Iron Enzyme AurF” J. Am. Chem. Soc. 2017, 139, 7062-7070. [link to publisher’s website] Peck, S. C., Wang, C.; Dassama, L. M. K.; Zhang, B.; Guo, Y.; Rajakovich, L. J.; Bollinger, J. M., Jr.; Krebs, C.; van der Donk, W. A. “O-H Activation by an Unexpected Ferryl Intermediate during Catalysis by 2-Hydroxyethylphosphonate Dioxygenase” J. Am. Chem. Soc. 2017, 139, 2045-2052. [link to publisher’s website] Martinie, R. J.; Blaesi, E. J.; Krebs, C.; Bollinger, J. M., Jr.; Silakov, A.; Pollock, C. J. “Evidence for a Di-µ-oxo Diamond Core in the Mn(IV)/Fe(IV) Activation Intermediate of Ribonucleotide Reductase from Chlamydia trachomatis” J. Am. Chem. Soc. 2017, 139, 1950-1957. [link to publisher’s website]