ENZYMOLOGY OF 2-OXOGLUTARATE-DEPENDENT DIOXYGENASES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1000973
• Project Start Date: Nov 1, 2013
• Project End Date: Oct 31, 2018
• Program Code: [(N/A)]- (N/A)

Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824

Performing Department
Microbiology & Molecular Genetics

Non Technical Summary
Enzymes known as 2-oxoglutarate (2OG)-dependent dioxygenases catalyze a wide range of chemical transformations that are important to agriculture. For example, agriculturally-signficant animals utilize such enzymes during synthesis of the critical structural protein collagen, to degrade phytanic acid that is present in plant-derived substances, and as a DNA repair mechanism. Plants synthesize an array of metabolites, including some antioxidants for human consumption, by use of these enzymes. Some pathogens contain specific examples of these enzymes that might be useful targets for inhibiting their growth. Several types of antibiotics include these enzymes in their biosynthetic pathways. Selected microbes contain representatives of these enzymes that degrade herbicides or other xenobiotic molecules. The full range of activities attributed to this enzyme family remains to be defined, and the detailed catalytic mechanism (important for the design of inhbitiors) requires further analysis to identify and characterize chemical intermediates. By careful analysis of appropriate model enzymes by using a range of biophysical approaches we will further clarify the precise steps in the overall chemical reactions of these enzymes. In addition, we will explore the functions of currently uncharacterized representatives of this enzyme family.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	4099	1000	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4099 - Microorganisms, general/other;

Field Of Science
1000 - Biochemistry and biophysics;

Keywords

dioxygenase

2-oxoglutarate

spectroscopy

enzymology

Goals / Objectives
Members of the 2-oxoglutarate-dependent dioxygenase enzyme family catalyze an amazing diversity of reaction, many of which are important to agriculture. The goals of this project are (1) to further dissect the mechanism of representative members of this group of enzymes, including the identification and characterization of catalytic intermediates, and (2) to uncover the functions of selected genes that encode new members of this enzyme family.

Project Methods
The archetype member of the 2-oxoglutarate-dependent dioxygenases, the taurine-degrading enzyme TauD and its variants, will be purified by established methods and further characterized by a battery of spectroscopic and other biophysical approaches. These approaches will include ultaviolet-visible (UV-vis) spectroscopy under anaerobic conditions (where the presence of 2-oxoglutarate generates a chromophore), stopped-flow UV-vis spectroscopy (for analysis of intermediates), fluorescence spectroscopy (to assess changes in endogenous Trp residue environments upon binding of ligands), thin-layer cyclic voltammetry, spectroelectrochemistry (to assess the reduction potential), and electron spin-echo envelope modulation (ESEEM) spectroscopy and hyperfine sub-level correlation (HYSCORE) spectroscopy of its nitric oxide-bound forms (thus creating a paramagnetic state for analysis of the environment), and cryogenic continuous-flow resonance Raman spectroscopy (to assess transient intermediates). These methods, some of which will be done in collaboration with the McCracken and Proshlyakov labs and all of which have already been used by us, will better define the intermediates in catalysis and the environment of the metallocenter for various states of the enzyme. A thermophilic paralog of TauD will be studied, such as the gene from Mycobacterium thermoresistibile, to allow use of a different temperature regime that may allow visualization of catalytic intermediates without the need for cryosolvents. Other model enzyme systems may also be examined, potentially requiring the cloning of the appropriate genes, purification of recombinant proteins, analysis of the isolated enzyme by routine biochemical procedures, and analysis of catalytic intermediates by the methods described above. Selected open reading frames of potential interest (because they are likely to encode members of this enzyme family from key host cells or suspected of association with a transformation activity of interest) will be cloned and the recombinant proteins purified. Direct tests of suspected activity (e.g., based on neighboring genes of known activity) will be carried out. If the reaction is identified, kinetic analysis will be carried out and the substrate specificity will be refined. When the function cannot be discerned readily, knockout strains of the native host will be developed and phenotypically characterized. In addition, mass spectrometric and other approaches will be used to define changes in metabolites within or secreted from these strains compared to the wild-type microbes. In other cases, BioLog phenotyping plates and other approaches will be utlized to define the function associated with the open reading frame.

Progress 11/01/13 to 10/31/18

Outputs
Target Audience:Scientists working in the fields of bioinorganic chemistry, enzymology, microbiology, and other areas. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In addition to the actual research work providing broad-based laboratory training for molecular biological methods and analysis of metalloproteins, our group meetings help to develop presentation skills and keep up with the literature. One-on-one sessions between researchers and the PI are held weekly to review data, plan experiments, and discuss ancillary topics. Lab personnel also participate in a biweekly discussion group entitled Metals, Membranes, and Metabolism to broaden their expertise as well as departmental seminars in microbiology, biochemistry, or chemistry. Professional development opportunities range from workshops on responsible conduct of research to student-learning methods to certification in college teaching. Some lab personnel attended scientific conferences. How have the results been disseminated to communities of interest?In addition to publishing the results of our studies, we have presented this work in oral and/or poster format at scientific conferences and meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We provided new insights into the diversity and functions of ferrous ion and 2-oxoglutarate dependent oxygenases by focusing on 5 projects. (1) We extended our exploration of the properties of the TauD sulfonate-degrading enzyme, the paradigm of the enzyme family, by examining its global stability in the presence of its substrates. In addition, we reviewed the spectroscopy of TauD as a case study for this entire enzyme family. (2) We combined our work on protein purification and site-directed mutagenesis studies with bioinformatics and molecular modeling efforts to identify a link between ALKBH7, a 2-oxoglutarate dependent oxygenase of unknown function, and prostate cancer. (3) We characterized the AP lyase and 6-methyl adenine demethylase activities of human ALKBH1. In addition, we used CRISPR/Cas9 technology to delete portions of three exons in the mammalian ALKBH1 demethylase/AP lyase to explore its in vivo role. (4) We continued our characterization of E. coli protein CsiD that hydroxylates glutaric acid as a substrate during late stationary phase growth, thus linking this enzyme to the L-hydroxyglutarate oxidase which is encoded by an adjacent gene. (5) We purified and extensively characterized a recombinant form of the ethylene-forming enzyme (EFE) from Pseudomonas and showed that its two activities, L-Arg hydroxylation and ethylene formation, can be unlinked. We additionally obtained 11 crystal structures of EFE in the presence of various substrates and substrate analogs, and reported the ethylene forming and L-Arg hydroxylation activities of ~30 variant forms of the protein. In addition, we carried out a series of thermodynamic analyses of this protein in the presence of various bound ligands. We also investigated an ethylene-forming enzyme from Penicillin digitalum and showed that it exhibited a greater ratio of ethylene produced per L-Arg hydroxylated than the Pseudomonas enzyme.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: M�ller, T. A., S. L. Struble, K. Meek, and R. P. Hausinger. 2018. Characterization of human AlkB homolog 1 produced in mammalian cells and demonstration of mitochondrial dysfunction in ALKBH1-deficient cells. Biochem. Biophys. Res. Commun. 495:98-103.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Herr, C. Q, and R. P. Hausinger. 2018. Amazing diversity in biochemical roles of Fe(II)/2-oxoglutarate oxygenases. Trends Biochem. Sci. 32:517-532.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Li, M., K. L. Henderson, S. Martinez, R. P. Hausinger, and J. P. Emerson. 2018. The Irving-Williams series and the 2-His-1-carboxylate triad: A thermodynamic study of Mn2+, Fe2+, and Co2+ binding to taurine ?-ketoglutarate dioxygenase (TauD). J. Biol. Inorg. Chem. 23:785-793.
• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Li, M., S. Martinez, R. P. Hausinger, and J. P. Emerson. 2018. Thermodynamics of iron(II) and substrate binding to the ethylene-forming enzyme. Biochemistry, in press

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Scientists working in the fields of bioinorganic chemistry, enzymology, microbiology, and other areas. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In addition to the actual research work providing broad-based laboratory training for molecular biological methods and analysis of metalloproteins, our group meetings help to develop presentation skills and keep up with the literature. One-on-one sessions between researchers and the PI are held weekly to review data, plan experiments, and discuss ancillary topics. Lab personnel also participate in a biweekly discussion group entitled Metals, Membranes, and Metabolism to broaden their expertise as well as departmental seminars in microbiology, biochemistry, or chemistry. Professional development opportunities range from workshops on responsible conduct of research to student-learning methods to certification in college teaching. Some lab personnel attended scientific conferences. How have the results been disseminated to communities of interest?In addition to publishing the results of our studies, we have presented this work in oral and/or poster format at scientific conferences and meetings. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue efforts to better understand the chemical mechanism of this important family of enzymes by using a variety of biophysical approaches. We will further examine the kinetics and spectroscopy associated with CsiD. Additional variants of EFE will be created and altered forms of the enzyme will be kinetically and structurally characterized. Manuscripts describing the results of this work will be prepared.

Impacts
What was accomplished under these goals? We continued to provide new insights into the diversity and functions of ferrous ion and 2-oxoglutarate dependent oxygenases by focusing on 5 projects. (1) We extended our exploration of the properties of the TauD sulfonate-degrading enzyme, the paradigm of the enzyme family, by examining its global stability in the presence of its substrates. In addition, we reviewed the spectroscopy of TauD as a case study for this entire enzyme family. (2) We combined our work on protein purification and site-directed mutagenesis studies with bioinformatics and molecular modeling efforts to identify a link between ALKBH7, a 2-oxoglutarate dependent oxygenase of unknown function, and prostate cancer. (3) We characterized the AP lyase and 6-methyl adenine demethylase activities of human ALKBH1. In addition, we used CRISPR/Cas9 technology to delete portions of three exons in the mammalian ALKBH1 demethylase/AP lyase to explore its in vivo role. (4) We continued our characterization of E. coli protein CsiD that hydroxylates glutaric acid as a substrate during late stationary phase growth, thus linking this enzyme to the L-hydroxyglutarate oxidase which is encoded by an adjacent gene (manuscript in preparation). (5) We purified and extensively characterized a recombinant form of the ethylene-forming enzyme (EFE) from Pseudomonas and showed that its two activities, L-Arg hydroxylation and ethylene formation, can be unlinked. We additionally obtained 11 crystal structures of EFE in the presence of various substrates and substrate analogs, and reported the ethylene forming and L-Arg hydroxylation activities of ~30 variant forms of the protein.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Martinez, S., and R. P. Hausinger. 2016. Biochemical and spectroscopic characterization of the non-heme Fe(II)- and 2-oxoglutarate-dependent ethylene-forming enzyme from Pseudomonas syringae pv. phasiolicola PK2. Biochemistry 55:5989-5999.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Proshlyakov, D. A., J. McCracken, and R. P. Hausinger. 2017. Spectroscopic analyses of 2-oxoglutarate-dependent oxygenases: TauD as a case study. J. Biol. Inorg. Chem. 22:367-379.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Henderson, K. L., M. Li, S. Martinez, E. A. Lewis, R. P. Hausinger, and J. P. Emerson. 2017. Global stability of an ?-ketoglutarate-dependent dioxygenase (TauD) and its related complexes. Biochim. Biophys. Acta 1861:987-994.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Walker, A. R., P. Silvestrov, T. A. M�ller, R. H. Podolski, G. Dyson, R. P. Hausinger, and G. A. Cisneros. 2017. ALKBH7 variant related to prostate cancer exhibits altered substrate binding. PLoS Comput. Biol. 13:e1005345.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: M�ller, T. A., M. Tobar, M. N. Perian, and R. P. Hausinger. 2017. Biochemical characterization of AP lyase and m6A demethylase activities of human AlkB homologue 1 (ALKBH1). Biochemistry 56:1899-1910.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Martinez, S., M. Fellner, C. Q Herr, A. Ritchie, J. Hu, and R. P. Hausinger. 2017. Structures and mechanisms of the non-heme Fe(II)- and 2-oxoglutarate-dependent ethylene-forming enzyme: Substrate binding creates a twist. J. Am. Chem. Soc., 139:11980-11988.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Scientists working in the fields of bioinorganic chemistry, enzymology, microbiology, and other areas. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In addition to the actual research work providing broad-based laboratory training for molecular biological methods and analysis of metalloproteins, our weekly group meetings help to develop presentation skills and keep up with the literature. One-on-one sessions between researchers and the PI are held weekly to review data, plan experiments, and discuss ancillary topics. Lab personnel also participate in a biweekly discussion group entitled Metals, Membranes, and Metabolism to broaden their expertise as well as departmental seminars in microbiology, biochemistry, or chemistry. Professional development opportunities range from workshops on responsible conduct of research to student-learning methods to certification in college teaching. Some lab personnel attended scientific conferences. How have the results been disseminated to communities of interest?In addition to submitting several manuscripts for publication, we have presented this work in oral and/or poster format at scientific conferences and meetings. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue efforts to better understand the chemical mechanism of this important family of enzymes by using a variety of biophysical approaches. We will define the site of covalent linkage between ALKBH1 and its lyase cleavage product, and we will examine the role of this enzyme in the mitochondria. We will extend our studies of the ethylene-forming enzyme to orthologues from other species and to site-directed variants in order to understand the residues important for its two activities. We will further examine the kinetics of the reaction catalyzed by CsiD. Manuscripts describing much of the above work along with other prior studies will be prepared.

Impacts
What was accomplished under these goals? Our studies have continued to provide new insights into the diversity and functions of ferrous ion and 2-oxoglutarate dependent oxygenases. We have extended our exploration of the properties of the TauD sulfonate-degrading enzyme, the paradigm of the enzyme family, by examining its global stability in the presence of its substrates (manuscript submitted). We also have reviewed the spectroscopy of this enzyme family using TauD as the case study (manuscript submitted). We combined our work on protein purification and site-directed mutagenesis studies with bioinformatics and molecular modeling efforts to identify a link between ALKBH7, a 2-oxoglutarate dependent oxygenase of unknown function, and prostate cancer (manuscript submitted). In addition, we used CRISPR/Cas9 technology to delete portions of three exons in the mammalian ALKBH1 demethylase/AP lyase to explore its in vivo role. We have continued our characterization of E. coli protein CsiD that hydroxylates glutaric acid as a substrate during late stationary phase growth, thus linking this enzyme to the L-hydroxyglutarate oxidase which is encoded by an adjacent gene. We have purified and extensively characterized a recombinant form of the ethylene-forming enzyme from Pseudomonas and shown that its two activities, L-Arg hydroxylation and ethylene formation, can be unlinked (manuscript submitted).

Publications

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Scientists working in the fields of bioinorganic chemistry, enzymology, microbiology, and other areas. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In addition to the actual research work providing broad-based laboratory training for molecular biological methods and analysis of metalloproteins, our weekly group meetings help to develop presentation skills and keep up with the literature. One-on-one sessions between researchers and the PI are held weekly to review data, plan experiments, and discuss ancillary topics. Lab personnel also participate in a biweekly discussion group entitled Metals, Membranes, and Metabolism to broaden their expertise as well as departmental seminars in microbiology, biochemistry, or chemistry. Professional development opportunities range from workshops on responsible conduct of research to student-learning methods to certification in college teaching. Some lab personnel attended scientific conferences. How have the results been disseminated to communities of interest?In addition to the published book and journal articles, we have presented this work in oral and/or poster format at scientific conferences and meetings. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue efforts to better understand the chemical mechanism of this important family of enzymes by probing TauD and one of its thermophilic homologs by using a variety of biophysical approaches. We will define the site of covalent interaction between ALKBH1 and its lyase cleavage product, and we will examine the role of this enzyme in the mitochondria. We will continue examining the ethylene-forming enzyme to establish the stoichiometries of substrates and products and we will expand in a new directly to test for reaction intermediates by stopped-flow UV-visible spectroscopy. We will further examine the kinetics of the reaction catalyzed by CsiD. Manuscripts describing much of the above work along with other prior studies will be prepared.

Impacts
What was accomplished under these goals? Our studies have provided new insights into the diversity and functions of ferrous ion and 2-oxoglutarate dependent oxygenases. We edited a book on this subject that unites the field by bringing together into a single source a review of the disparate activities of these enzymes including animal-derived examples used for DNA repair, hypoxic signaling, collagen synthesis, and many other needs; plant-derived enzymes for biosynthesis of flavonoids, gibberillins, and alkaloids; and microbial enzymes that decompose herbicides and other compounds or synthesize antibiotics and other commodities. In addition to contributing two of the 21 chapters in this book, we prepared a separate review of the enzyme mechanisms of this oxygenase family. We have continued to explore the properties of the TauD sulfonate-degrading enzyme, the paradigm of the enzyme family, especially with regard to its thermodynamics of binding iron and substrates. We combined molecular modeling with site-directed mutagenesis studies to test whether particular features of the ALKBH1 demethylase/lyase are critical to its function. In addition, we used CRISPR/Cas9 technology to delete portions of three exons in the mammalian enzyme to explore its in vivo role. We demonstrated that the E. coli protein, CsiD, hydroxylates glutaric acid as a substrate during late stationary phase growth, thus linking this enzyme to the L-hydroxyglutarate oxidase which is encoded by an adjacent gene. An improved purification procedure was developed for a recombinant form of the ethylene-forming enzyme from Pseudomonas and the activity was kinetically characterized. Arginine and 2-oxoglutarate analogues were examined as alternative inducers and substrates.

Publications

• Type: Book Chapters Status: Published Year Published: 2015 Citation: M�ller, T. A., and R. P. Hausinger. 2015. AlkB catalyzed DNA/RNA repair and related topics. In 2-Oxoglutarate-Dependent Oxygenases (Eds. Schofield, C. J., and R. P. Hausinger), RSC Publishing, Royal Society of Chemisty, Cambridge, U.K., pp.246-262.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Silvestrov, P., T. A. M�ller, K. N. Clark, R. P. Hausinger, and G. A. Cisneros. 2014. Homology modeling, molecular dynamics, and site-directed mutagenesis study of AlkB human homolog 1 (ALKBH1). J. Molec. Graph. Mod. 54:123-130, PMC4268226.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Martinez, S., and R. P. Hausinger. 2015. Catalytic mechanism of Fe(II)- and 2-oxoglutarate-dependent oxygenases. J. Biol. Chem. 290:20702-20711. PMC4543632.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Henderson, K. L., T. A. M�ller, R. P. Hausinger, and J. P. Emerson. 2015. A calorimetric assessment of Fe2+ binding to alpha-ketoglutarate/taurine dioxygenase: Ironing out the energetics of metal coordination by the 2-His-1-carboxylate facial triad. Inorg. Chem. 54:2278-2283.
• Type: Books Status: Published Year Published: 2015 Citation: Schofield, C. J. and R. P. Hausinger (Eds.). 2015. 2-Oxoglutarate-Dependent Oxygenases, 487 p., RSC Publishing, Royal Society of Chemisty, Cambridge, U.K.
• Type: Book Chapters Status: Published Year Published: 2015 Citation: Hausinger, R. P. 2015. Biochemical diversity of 2-oxoglutarate-dependent oxygenases. In 2-Oxoglutarate-Dependent Oxygenases (Eds. Schofield, C. J., and R. P. Hausinger), RSC Publishing, Royal Society of Chemisty, Cambridge, U.K., pp.1-58.

Progress 11/01/13 to 09/30/14

Outputs
Target Audience: Scientists working in the fields of bioinorganic chemistry, enzymology, microbiology, and other areas. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? In addition to the actual research work providing broad-based laboratory training for molecular biological methods and analysis of metalloproteins, our weekly group meetings help to develop presentation skills and keep up with the literature. One-on-one sessions between researchers and the PI are held weekly to review data, plan experiments, and discuss ancillary topics. Lab personnel also participate in a biweekly discussion group entitled Metals, Membranes, and Metabolism to broaden their expertise as well as departmental seminars in microbiology, biochemistry, or chemistry. Professional development opportunities range from workshops on responsible conduct of research to student-learning methods to certification in college teaching. Some lab personnel attended scientific conferences. How have the results been disseminated to communities of interest? The results of this work were presented in oral and/or poster format at scientific conferences and meetings, and the work was highlighted in seminars at other institutions. What do you plan to do during the next reporting period to accomplish the goals? We plan to continue efforts to better understand the chemical mechanism of this important family of enzymes by probing TauD and its homologs using a variety of biophysical approaches. Of particular interest is a potential thermophilic version that we will study as soon as we receive the DNA. We will continue examining the ethylene-forming enzyme and establish the stoichiometries of substrates and products. We will establish the product of the reaction catalyzed by CsiD. Manuscripts describing much of the above work along with other prior studies will be prepared. A book entitled “2-Oxoglutarate-Dependent Oxygenases” and co-edited by the PI will be published by the Royal Society for Chemistry.

Impacts
What was accomplished under these goals? Our studies have provided new insights into the diversity and functions of ferrous ion and alpha-ketoglutarate dependent dioxygenases and related proteins. Representative family members possess a variety of roles, many of which are significant to agriculture (e.g., biosynthesis of plant products such as flavonoids, gibberillins, and alkaloids; animal enzymes used for DNA repair, hypoxic signaling, collagen synthesis, and many other needs; and microbial enzymes that decompose herbicides and other compounds or synthesize antibiotics and other commodities). The TauD sulfonate-degrading system continues to serve as the paradigm of the enzyme family, especially with regard to elucidation of the catalytic mechanism. Using TauD, we carried out isothermal titration calorimetry studies with a collaborator who is expert in this approach. Such measurements have not been reported for any other member of the Fe(II)/alpha-ketoglutarate-dependent oxygenase family. We attempted to obtain TauD homologs from thermophilic microorganisms for detailed mechanistic studies, but this has been unsuccessful thus far due to insolubility, inactivity, or lack of thermal stability. One human homologue of the DNA repair protein AlkB, known as ALKBH1, was localized to the mitochondrion—providing important information into its function. An E. coli protein, CsiD, of undefined function was shown to utilize trans-glutaconic acid as a substrate during late stationary phase growth. A recombinant form of the ethylene-forming enzyme from Pseudomonas was kinetically characterized and mechanistic studies have been initiated. Evidence for self-hydroxylation of the protein was obtained by spectrophotometric methods.

Publications