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Principal Investigator: Yisong Guo
Organization: CARNEGIE-MELLON UNIVERSITY
Fiscal Year: 2024
Award: $341,407
Funding agency: National Institute of General Medical Sciences
Project Summary/Abstract
Iron and 2-oxoglutarate-dependent (Fe/2OG) enzymes, representing a superfamily of non-heme mononuclear
iron-containing (NHM-Fe) enzymes, have garnered strong research interests from fundamental enzyme
mechanism studies to bioengineering/biocatalysis explorations in recent years due to their exceedingly diverse
catalytic reactivities and simple enzyme architectures. Radical halogenation reactions via C-H bond activation
catalyzed by Fe/2OG halogenases are particularly attractive for chemical synthesis and biocatalysis applications,
since these enzymes can install carbon-halide bonds in a regio- and stereo-specific manner, a feat that has yet
to be achieved by organic synthetic methodology. As revealed by the mechanistic studies of carrier protein-
dependent Fe/2OG halogenases, the key step in the radical halogenation mechanism is the selective halide
radical transfer from the hydroxo-Fe(III)-halide intermediate to the substrate radical generated by the key reactive
species, the ferryl (Fe(IV)=O) intermediate. However, a consensus mechanism to explain the selective halide
transfer in Fe/2OG halogenases has not been reached, particularly the controlling factors to avoid hydroxyl
radical transfer to lead to hydroxylation reaction are not fully revealed. Additionally, the reasons why Fe/2OG
enzymes cannot perform fluorination reaction are completely unknown. In this project, we will bridge these
knowledge gaps by studying two newly discovered carrier protein-independent Fe/2OG halogenases that
catalyze chlorination reactions to generate halogenated nucleotide natural products and halogenated free-
standing amino acids. By using an integrative approach consisting of mechanistic probe design and synthesis,
enzyme product structural determination via LC-MS and NMR analysis, transient enzyme kinetics, advanced
spectroscopic characterization and molecular dynamics simulations, we will elucidate the influence of protein-
substrate interactions and dynamics in controlling efficient halogenation, explore the effect of different iron-bound
anions (e.g. Cl- vs. F-) to the electronic structure and the reactivity of the ferryl intermediate, test new chemical
strategies to enable fluorination in Fe/2OG enzymes, and expand the substrate scope of these enzymes for
potential synthetic applications. Given the importance of halogen-containing organic molecules in the modern
pharmaceutical and agrochemical applications, mechanistic elucidation of these newly discovered halogenases
will lay scientific foundation for future biocatalytic applications of these unique enzymes.
Terms: <2'-deoxyadenosine><2-ketoglutarate><2-oxoglutarate><Agricultural Chemicals><Agrochemicals><Alkenes><Alkynes><Amino Acids><Anabolism><Anions><Anti-Cancer Agents><Antineoplastic Agents><Antineoplastic Drugs><Antineoplastics><Architecture><Azides><Binding><Biomedical Engineering><Cancer Drug><Carbon><Carrier Proteins><Chemicals><Chlorides><Consensus><Copper><Cu element><Data><Electronics><Engineering / Architecture><Environment><Enzyme Gene><Enzyme Kinetics><Enzymes><Exhibits><Family><Fe element><Foundations><Future><GeneHomolog><Genetics-Mutagenesis><Grant><Group 17 Elements><H element><H-bond><Halogens><Homolog><Homologous Gene><Homologue><Hydrogen><Hydrogen Bonding><Hydroxyl><Hydroxyl Radical><Hydroxylation><Iron><Ketoquinolines><Knowledge><Leucine><Libraries><Methodology><Modernization><Molecular Dynamics Simulation><Molecular Interaction><Mononuclear><Mutagenesis><Mutagenesis Molecular Biology><Natural Products><Neoplastic Disease Chemotherapeutic Agents><Nucleosides><Nucleotide Biosynthesis><Nucleotides><O element><O2 element><Olefins><Outcome><Oxidants><Oxidizing Agents><Oxoquinolines><Oxygen><Pathway interactions><Pharmaceutical Agent><Pharmaceuticals><Pharmacologic Substance><Pharmacological Substance><Position><Positioning Attribute><Property><Proteins><Quinolinones><Quinolones><Race><Races><Reaction><Research><Science><Shapes><Site><Substrate Interaction><Testing><Transport Protein Gene><Transport Proteins><Transporter Protein><Tumor-Specific Treatment Agents><Viral><alpha ketoglutarate><alpha-oxoglutarate><aminoacid><anti-cancer drug><bio-engineered><bio-engineers><bioengineering><biological engineering><biosynthesis><chemical synthesis><chlorination><cycloaddition><design><designing><drug discovery><electron acceptor><electronic><electronic device><electronic structure><enzyme mechanism><enzyme substrate><fluorophore><geometric structure><halogenation><insight><interest><molecular dynamics><naturally occurring product><novel><pathway><pharmaceutical><racial><racial background><racial origin><screening><screenings><spectroscopic study><spectroscopic survey><structural geometry><synthetic drug><α-ketoglutarate><α-oxoglutarate><αKG>