Progress 11/01/08 to 10/31/13
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 helped to develop presentation skills and keep up with the literature. One-on-one sessions between researchers and the PI were held weekly to review data, plan experiments, and discuss ancillary topics. Lab personnel also participated 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 ranged 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? Publications in scientific journals describing our primary research along with review articles. The results of this work also 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?
Nothing Reported
Impacts
What was accomplished under these goals?
Our studies provided new insights into the diversity of ferrous ion and alpha-ketoglutarate dependent dioxygenases and related proteins. The TauD system continues to serve as the paradigm of the enzyme family, especially with regard to elucidation of the catalytic mechanism. Other representative family members exhibit diverse functions, many of which are significant to agriculture. Using TauD, we carried out thin film cyclic voltammetry and UV-visible titration studies with oxidized and reduced electron carriers to assess the reduction potential of the active site. The electrochemical studies were ineffective, even when a mediator was used, while the UV-visible spectroscopy approach was successful in providing a midpoint potential. Such measurements have not been reported for any other member of the Fe(II)/alpha-ketoglutarate-dependent oxygenase family. Surprisingly, the addition of the substrates alpha-ketoglutarate and/or taurine had negligible effects on the reduction potential suggesting that this approach might only detect the holoprotein and cannot be used with reversibly bound ligands. We also applied electron paramagnetic resonance (EPR) and electron spin-echo envelopment modulation (ESEEM, a pulsed EPR method) spectroscopies to TauD, with improved methods devised for simulating the spectra. Furthermore, a two-dimensional analysis known as HYSCORE (hyperlevel sublevel correlation) spectroscopy that provides more refined insights was utilized with this enzyme. A human homologue of AlkB, known as ALKBH1, that cleaves abasic sites in DNA was shown to covalently attach to the 5’ product. The site of cross-linkage was localized to the amino terminal 64 residues whereas Lys 133 appears to be the key residue for catalysis of the lyase chemistry. We recreated and characterized a strain of ALKBH1-deficient mice (which exhibits reduced survival rates and non-Mendelian inheritance), and generated stable fibroblast cell lines along with cells expressing the corresponding human gene. The splenic B cells from knockout mice were used demonstrate that ALKBH1 is dispensable for class switch recombination, part of the process used by cells to generate antibody diversity. Similarly, the tissue culture cells were utilized to show that ALKBH1 is not required for repair of DNA damage from exposure to hydrogen peroxide or methyl methanesulfonate. Publications in scientific journals describing our primary research along with review articles. Patent applications also are possible. The results of this work will be presented in oral and/or poster format at scientific conferences and meetings, and the work will be highlighted in seminars at other institutions. (Prior progress reports describe accomplishments in earlier years).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
M�ller, T. A., Andrzejak, M. M., Hausinger, R. P. 2013. A covalent protein-DNA 5-product is generated following AP lyase activity of human ALKBH1 (AlkB homologue 1). Biochem. J. 452:509-518.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
M�ller, T. A., Yu, K., Hausinger, R. P., Meek, K. 2013. ALKBH1 is dispensable for abasic site cleavage during base excision repair and class switch recombination. PLoS One 8:e67403.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Casey, T. M., Grzyska, P. K., Hausinger, R. P., McCracken, J. 2013. Measuring the orientation of taurine in the active site of the non-heme Fe(II)/alpha-ketoglutarate-dependent taurine hydroxlase (TauD) using electron spin echo envelope modulation (ESEEM). J. Phys. Chem. B 117:10384-10394.
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2013
Citation:
Warrell, C. A. 2013. Measuring the reduction potential of taurine:alpha-ketoglutarate dioxygenase and biochemical analysis of a thermophilic ortholog
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: This project focuses on the characterization of several ferrous ion and alpha-ketoglutarate dependent dioxygenases that decarboxylate alpha-ketoglutarate while hydroxylating their primary substrate targets. We continue to characterize the prototype of this enzyme family, TauD, a taurine-degrading enzyme. Mechanistically related enzymes that were investigated include AlkB homologues involved in DNA repair and other roles from a Trypanosome and from humans, along with preliminary efforts involving a 5-methylcytosine demethylase and a methyl-lysine histone demethylase. The work was presented as oral presentations at several scientific meetings and some of the results have been published. PARTICIPANTS: PI: Robert P. Hausinger; Partner organizations, none; Collaborators: Denis Proshlyakov, John McCracken, Donna Koslowsky, and Kathy Meek; Training or professional development: Post-doctoral scientists included Tina Mueller, Scott Mulrooney, and Jana Simmons; graduate students included Celeste Warrell and Thomas Casey (the latter with McCracken). TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The studies described here provide new insights into the diversity of ferrous ion and alpha-ketoglutarate dependent dioxygenases and related proteins. The TauD system continues to serve as the paradigm of the enzyme family, especially with regard to elucidation of the catalytic mechanism. Other representative family members exhibit diverse functions, many of which are significant to agriculture. Using TauD, we carried out thin film cyclic voltammetry and UV-visible titration studies with oxidized and reduced electron carriers to assess the reduction potential of the active site. We applied electron paramagnetic resonance (EPR) and electron spin-echo envelopment modulation (ESEEM, a pulsed EPR method) spectroscopies to TauD, with improved methods devised for simulating the spectra. An AlkB homologue from Trypanosoma brucei was demonstrated to confer some protection against alkylating agents to an alkB mutant of E. coli, consistent with a protective role in demethylating alkylation lesions. A human homologue of AlkB, known as ALKBH1, that cleaves abasic sites in DNA was shown to covalently attach to the 5' product. Efforts to obtain a soluble domain of Tet1, a 5-methylcytosine demethylase, for spectroscopic and mechanistic studies were unsuccessful. By contrast, we were successful in expressing two human histone demethylases and have begun to work out a purification protocol.
Publications
- Simmons, J. M., Koslowsky, D. J., Hausinger, R. P. 2012. Characterization of a Trypanosoma brucei AlkB homolog capable of repairing alkylated DNA. Exp.Parasitol. 131:92-100.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: This project focuses on the characterization of several ferrous ion and alpha-ketoglutarate dependent dioxygenases that decarboxylate alpha-ketoglutarate while hydroxylating their primary substrate targets. Some effort involved characterization of the prototype of this enzyme family, TauD, a taurine-degrading enzyme. Mechanistically related enzymes that were investigated include XanA, a xanthine-degrading enzyme, two AlkB homologues from a Trypanosome and from humans, involved in DNA repair and other roles, and a flavoenzyme, AidB. The work was presented as oral presentations at several scientific meetings and some of the results have been published. PARTICIPANTS: PI: Robert P. Hausinger; Partner organizations, none; Collaborators: Denis Proshlyakov, John McCracken, Donna Koslowski, and Kathy Meek; Training or professional development: Post-doctoral scientists included Tina Mueller, William Kittleman, Scott Mulrooney, and Jana Simmons; graduate students included Thomas Casey (with McCracken). TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The studies described here provide new insights into the diversity of ferrous ion and alpha-ketoglutarate dependent dioxygenases and related proteins. The TauD system continues to serve as the paradigm of the enzyme family, especially with regard to elucidation of the catalytic mechanism. Other representative family members exhibit diverse functions, many of which are significant to agriculture. We showed TauD binds a second Fe(II) at a site that is distant from its substrate binding site; however, on the basis of mutagenesis experiments that disrupt the second site, it has only modest effects on TauD activity. Electron paramagnetic resonance (EPR) and electron spin-echo envelopment modulation (ESEEM, a pulsed EPR method) spectroscopies were applied to TauD, with improved methods devised for simulating the spectra. Additional ESEEM studies were carried out with nitric oxide-bound XanA. A gene encoding an analogue of AlkB, a DNA repair enzyme, previously was cloned by us from Trypanosoma brucei and expressed in Escherichia coli. The recombinant protein was purified and characterized for binding Fe(II) and alpha-ketoglutarate, and the gene was shown to confer some protection against alkylating agents to E. coli. A human homologue of AlkB, known as ABH1, was shown to bind covalently and cleave abasic sites in DNA, with the protein remaining bound to one product. AidB, a protein whose expression coincides with AlkB, was characterized and shown to be a redox partner of flavodoxin, to bind to RNA, and to bind to the acyl carrier protein. The function of the protein remains unclear.
Publications
- Proshlyakov, D. A., Hausinger, R. P. 2011. Transient iron species in the catalytic mechanism of the archetypal alpha-ketoglutarate-dependent dioxygenase, TauD. In Iron-Containing Enzymes: Versatile Catalysts of Hydroxylation Reactions in Nature (Eds., de Visser, S. and D. Kumar). Royal Society of Chemistry, Cambridge, U.K., pp. 67-87.
- Mulrooney, S. B., Howard, M. J., Hausinger, R. P.. 2011. The Escherichia coli alkylation response protein AidB is a redox partner of flavodoxin and binds RNA and acyl carrier protein. Arch. Biochem. Biophys. 513:81-86.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: This project focuses on the characterization of several ferrous ion and alpha-ketoglutarate dependent dioxygenases that decarboxylate alpha-ketoglutarate while hydroxylating their primary substrate targets. The prototype of this enzyme family is TauD, a taurine-degrading enzyme that we have extensively characterized by using biochemical, mutagenesis, and spectroscopic approaches. Using TauD, we obtained thermodynamic information related to Fe(II) binding by use of difference UV spectroscopy. Of greater importance, continuous flow resonance Raman studies of the protein revealed evidence for two new catalytic intermediates that were tentatively identified as Fe(III)-oxo and Fe(II)-alkoxo species. Surprisingly, the TauD protein was shown to possess a second Fe(II) binding site that is distant from its substrate binding site. Mutagenesis experiments suggest the second site does have some influence on TauD activity. Electron paramagnetic resonance (EPR) and electron spin-echo envelopment modulation (ESEEM, a pulsed EPR method) spectroscopies showed two nitric oxide binding modes to the Fe(II) site of XanA, a xanthine-degrading enzyme. Preliminary hyperfine sublevel correlation spectroscopy (HYSCORE, a 2D pulsed EPR method) was carried out with this enzyme and this technique was used to obtain more detailed characterization of the TauD catalytic metallocenter. Genes encoding three members of this enzyme family were cloned from Trypanosoma brucei and expressed in Escherichia coli, the proteins were purified and characterized for binding Fe(II) and alpha-ketoglutarate, and tests were conducted to experimentally test hypotheses for their function. No activity was detected for two of the proteins, but the third appears to exhibit 1-methyladenosine and/or 3-methylcytosine demethylase activity according to its ability to partially compensate for an alkB deficient mutant strain of E. coli in protecting the cells against alkylation damage. The work was presented as oral presentations at several scientific meetings and some of the results have been published. PARTICIPANTS: PI: Robert P. Hausinger; Partner organizations, none; Collaborators: Denis Proshlyakov, John McCracken, Donna Koslowski, and Kathy Meek; Training or professional development: Post-doctoral scientists included Tina Mueller, Piotr Grzyska, William Kittleman, Scott Mulrooney; graduate students included Jana Simmons; undergraduate students included Megan Andrzejak and Michael Howard. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The studies described here provide new insights into the diversity of ferrous ion and alpha-ketoglutarate dependent dioxygenases and related proteins. The TauD system continues to serve as the paradigm of the enzyme family, especially with regard to elucidation of the catalytic mechanism. Other representative family members exhibit diverse functions, many of which are significant to agriculture.
Publications
- Mueller, T. A., Meek, K., Hausinger, R. P. 2010. Human AlkB homologue 1 (ABH1) exhibits DNA lyase activity at abasic sites. DNA Repair 9:58-65. Grzyska, P. K., Hausinger, R. P., Proshlyakov, D. A. 2010. Metal and substrate binding to an Fe(II) dioxygenase resolved by UV spectroscopy with global regression analysis. Anal. Biochem. 399:64-71.
- Simmons, J. M., Koslowsky, D. J., Hausinger, R. P. 2010. Trypanosoma brucei brucei: Thymine 7-hydroxylase-like proteins. Exp. Parasitol. 124:453-458.
- Grzyska, P. K., Appelmann, E. H., Hausinger, R. P., Proshlyakov, D. A. 2010. Insight into the mechanism of an iron dioxygenase by resolution of steps following the Fe(IV)=O species. Proc. Natl. Acad. Sci. USA 107:3982-3987.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: This project focuses on the characterization of several ferrous ion and alpha-ketoglutarate dependent dioxygenases that decarboxylate alpha-ketoglutarate while hydroxylating their primary substrate targets. The prototype of this enzyme is TauD, a taurine degrading enzyme that has been extensively characterized by using biochemical, mutagenesis, and spectroscopic approaches. We have continued to explore this protein by using a variety of spectroscopic methods including electron spin-echo envelop modulation (ESEEM), hyperfine sublevel correlation (HYSCORE), continue flow resonance Raman, and difference UV absorption spectroscopies with our collaborators Dr. McCracken and Dr. Proshlyakov. In particular, we used nitric oxide bound (thus paramagnetic) species to probe the environment of the metallocenter in TauD and XanA, a mechanistically related xanthine-degrading enzyme. Furthermore, we carried out time resolved resonance Raman studies with taurine and its deuterated analogue to define the intermediates in TauD catalysis following the Fe(IV)-oxo species. Moreover, we showed that UV absorption can be a selective probe to monitor metal and substrate binding in TauD and, by implication, in a wide range of other proteins. We extensively characterized two thymine hydroxylase-like proteins from Trypanosoma brucei and confirmed they belong to the Fe(II)/alpha-ketoglutarate dioxygenase family even though we could not define their function. A protozoal AlkB-like protein also was characterized and shown to exhibit the ability to demethylate alkylated DNA. The human and mouse homologue 1 to AlkB was found to exhibit a lyase that is specific to abasic sites in addition to its dioxygenase activity. AidB, an enzyme that is co-expressed with AlkB, was examined by computational docking techniques to discern the types of compounds that are likely to bind to its active site. Three publications are now in press based on this work. PARTICIPANTS: PI: Robert P. Hausinger. Other workers: Dr. Tina Mueller, Dr. Piotr Grzyska, Dr. William Kittleman, Dr. Scott Mulrooney, graduate student Jana Simmons, and undergraduate students Megan Andrzejak, Michael Howard, and Rachel Morr. Partner organizations: none. Collaborators: Dr. Kathy Meek, Dr. Denis Proshlyakov, Dr. Donna Koslowsky, and Dr. John McCracken. Training or professional development: this pertains to those postdoctoral researchers and students listed under other workers. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The studies described here provide new insights into the diversity of ferrous ion and alpha-ketoglutarate dependent dioxygenases and related proteins. The TauD system continues to serve as the paradigm for elucidating the catalytic intermediates in this enzyme family. Other family members exhibit diverse functions, with some representatives being highly significant to agriculture.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: This project focuses on the characterization of several ferrous ion and alpha-ketoglutarate dependent dioxygenases that decarboxylate alpha-ketoglutarate while hydroxylating their primary substrate targets. The prototype of this enzyme family is TauD, a taurine degrading enzyme that we have extensively characterized by using biochemical, mutagenesis, and spectroscopic approaches. We have continued to explore this protein by use of a variety of spectroscopic methods including electron spin-echo envelop modulation (ESEEM), hyperfine sublevel correlation (HYSCORE), continuous flow resonance Raman, and difference UV-visible spectroscopies with our collaborators Dr. McCracken and Dr. Proshlyakov. In particular, we used the nitric oxide-bound (paramagnetic) species to probe the environment near the metallocenter center, further characterized the kinetics involving the Fe(IV)-oxo intermediate, and identified a new catalytic intermediate. We continued characterization of a xanthine metabolizing enzyme, XanA, by a site-directed mutagenesis approach. Two orthologues of genes encoding thymine 7-hydroxylase were cloned from Trypanosoma brucei and expressed in Escherichia coli, but no activity was detected. This protozoan also served as the source of a gene that resembles the DNA repair enzyme AlkB from E. coli. The protozoal orthologue was cloned and expressed in E. coli, and the protein was shown to possess DNA demethylase activity as measured by reduced toxicity of cells toward a methylating agent. The genes encoding human and mouse AlkB homologue 1 (ABH1) were cloned and expressed in E. coli. These proteins were shown to possess the unexpected capability of cleaving DNA at abasic sites. Surprisingly, the activity was not dependent on the presence of added Fe(II) or alpha-ketoglutarate, and variants affecting the metal binding ligands retained the endonuclease activity. The E. coli protein CsiD was previously crystallized by others, but not functionally characterized; we subjected this protein to a wide range of experimental studies and showed it to be a member of this enzyme family, yet its role remains unknown. In one effort to gain insight into the function of this protein, an adjacent co-transcribed gene of unknown role was cloned and the corresponding protein, YgaF, was purified, found to contain FAD, and shown to exhibit L-hydroxyglutarate oxidase activity. Finally, we have continued to characterize AidB, a protein that is co-regulated with AlkB and suspected to function in DNA repair. We showed the AidB flavoprotein forms tight complexes with the acyl carrier protein and with ferredoxin. PARTICIPANTS: PI: Robert P. Hausinger Partner organizations: none Collaborators: John McCracken, Denis Proshlyakov, Donna Koslowsky, and Kathy Meek Training or professional development: Post-doctoral scientists included Tina Mueller, Piotr Grzyska, and Scott Mulrooney; graduate students included Meng Li, Efthalia Kallliri, and Jana Simmons; undergraduate students included Megan Andrzejak, Bruce Fraser, Aaron Chester, and Greg Moyerbrailean. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The studies described here provide new insights into the diversity of ferrous ion and alpha-ketoglutarate dependent dioxygenases and related proteins. The TauD system continues to serve as the paradigm of this enzyme family, especially with regard to elucidation of catalytic intermediates. Other representative family members exhibit diverse functions, many of which are significant to agriculture.
Publications
- Li, M., Mueller, T. A., Fraser, B. A., Hausinger, R. P. 2008. Characterization of active site variants of xanthine hydroxylase from Aspergillus nidulans. Arch. Biochem. Biophys. 470:44-53.
- Kalliri, E., Mulrooney, S. B., Hausinger, R. P. 2008. Identification of Escherhichia coli YgaF as L-2-hydroxylglutarate oxidase. J. Bacteriol. 190:3793-3798.
- Simmons, J. M., Mueller, T. A., Hausinger, R. P. 2008. FeII/alpha-ketoglutarate hydroxylases involved in nucleobase, nucleoside, nucleotide, and chromatin metabolism. Dalton Trans. 38:5132-5142.
- Kalliri, E. 2008. Kinetic/spectroscopic investigation of TauD interactions with inhibitors and isolation/characterization of the putative Fe(II)/alpha-ketoglutarate dependent dioxygenase and flavin-dependent dehydrogenase CsiD and YgaF. Ph.D. thesis.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: This project focused on the characterization of several ferrous ion- and alpha-ketoglutarate dependent dioxygenases that oxidatively decarboxylate alpha-ketoglutarate while hydroxylating their primary substrate. In some cases, the hydroxylated compound is unstable and decomposes to the final products. We purified XanA from its natural host, Aspergillus nidulans, and as a recombinant enzyme from Escherichia coli. The two forms differed in several respects (native size, glycosylation, and phosphorylation), but exhibited similar kinetic parameters. The enzyme was shown to be an alpha-ketoglutarate dependent hydroxylase of xanthine, producing uric acid. The activity was specific to ferrous ion, with several other metals acting as competitive inhibitors. The cosubstrate could be substituted by alpha-ketoadipate and its use was inhibited by N-oxalylglycine. Dihydroxypurine was shown to be an inhibitor that competes with substrate. A homology model was created. We tested for
thymidine hydroxylase activity in a J-binding protein (JBP1) from Trypanosoma brucei. These protozoa are known to possess a modified DNA base, called J, and JBP1 was previously shown by others to bind to this base. Sequence analysis combined with examination of truncation mutants showed that only the C-terminal half of JBP1 is needed for DNA binding, and the N-terminal domain was shown to have features resembling alpha-ketoglutarate dependent dioxygenases. The purified recombinant protein isolated from E. coli did not exhibit any in vitro activity, however variant protein lacking the suspected metal ligands led to 20-fold reduction of J levels in trypanosomes. This work was carried out in collaboration with the Borst lab in the Netherlands. Several investigations were carried out with the best-studied representative of this enzyme class: TauD or taurine hydroxylase. Several variant proteins were created in which the three metal ligands were extensively substituted by other residues.
Analyses of these mutants led to the discovery that the active enzyme carries out aberrant chemistry in vivo, resulting in the self-hydroxylation of Tyr-73 with the dihydroxyphenylalanine subsequently undergoing oxidation to the orthoquinone. Further evidence for this posttranslational modification was obtained by incubation of anaerobic enzyme with chromium(II), which resulted in the formation of a chromium(III)-semiquinone species. The most important studies of TauD used electron spin echo envelope modulation (ESEEM) spectroscopy to probe the metallocenter environment in the presence of deuterated substrate (deuterated on the C1 carbon or uniformly labeled with the heavy isotope) and the oxygen analogue, nitric oxide (NO). In collaboration with Dr. John McCracken, an ESEEM method was developed to define the distance and orientation of the label from the Fe-NO center. Further development of this approach in two dimensions is in progress. In addition, we continue to work with Dr.
Denis Proshlyakov to apply continuous flow resonance Raman methods to this model enzyme.
PARTICIPANTS: PI: Robert P. Hausinger Partner Organizations: Institut de Genetique et de Microbiologie, Universite Paris-Sud; Universidad Nacional Autonoma de Mexico; John Innes Center, Norwich, UK; the Netherlands Cancer Institute, Amsterdam; Marine Biological Laboratory, Woods Hole, MA; University of Liverpool, Liverpool, UK. Collaborators: Michael Feig, John McCracken, Denis Proshlyakov. Training or professional development: Meng Li, Andrew Stumpff-Kane, Jana Simmons, Piotr Grzyska, Tina Mueller, Melody Campbell, Rajenda Bose Muthukumaran, Efthalia Kalliri, Aaron Chester, Bruce Fraser, Andrea Silva, Greg Moyerbrailean, Rachel Morr.
Impacts
The studies described here provide essential new insights into the diversity of ferrous ion- and alpha-ketoglutarate dependent dioxygenases and reveal important findings about their ability to catalyze self-hydroxylation reactions within the cell and the behavior of selected inhibitors. The TauD system was used to develop new spectroscopic approaches that can be used to examine other members of this widespread family of enzymes. Because related enzymes carry out a wide variety of agriculturally significant reactions, the studies carried out as part of this project have long range ramifications in understanding how to control and modify the properties of these critical catalysts.
Publications
- Muthukumaran, R. B., Grzyska, P.K., Hausinger, R.P., McCracken, J. 2007. Probing the Fe-substrate orientation for taurine/alpha-ketoglutarate dioxygenase using deuterium ESEEM spectroscopy. Biochemistry 46:5951-5959.
- Grzyska, P. K., Hausinger, R. P. 2007. Cr(II) reactivity of taurine/alpha-ketoglutarate dioxygenase. Inorg. Chem. 46:10087-10092.
- Li, M. 2007. Purification, kinetic analysis, and spectroscopic characterization of wild-type Aspergillus nidulans xanthine hydroxylase and selected variants. Ph.D. thesis.
- Montero-Moran, G.M., Li, M., Rendon-Huerta, E., Jourdan, F., Lowe, D.J., Stumpff-Kane, A.W., Feig, M., Scazzocchio, C., Hausinger, R.P. 2007. Purification and characterization of the Fe(II)- and alpha-ketoglutarate-dependent xanthine hydroxylase from Aspergillus nidulans. Biochemistry 46:5293-5304.
- Yu, Z., Genest, P.-A., ter Riet, B., Sweeney, K., DiPaolo, C., Kieft, R., Christodoulou, E., Perrakis, A., Simmons, J.M., Hausinger, R. P., van Luenen, H.G.A.M., Rigden, D.J., Sabatini, R., Borst, P. 2007. The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase. Nucl. Acids Res. 35:2107-2115.
- Grzyska, P. K., Mueller, T.A., Campbell, M.G., Hausinger, R.P. 2007. Metal ligand substitution and evidence for quinone formation in taurine/alpha-ketoglutarate dioxygenase. J. Inorg. Biochem. 101:797-808.
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Progress 01/01/06 to 12/31/06
Outputs
This project focused on the characterization of several ferrous ion- and alpha-ketoglutarate-dependent dioxygenases that hydroxylate their primary substrate while decomposing alpha-ketoglutarate to carbon dioxide plus succinate. We continued studies with the best-characterized representative of this enzyme family, TauD or taurine (2-aminoethanesulfonate) hydroxylase of E. coli, by focusing on kinetic and spectroscopic analysis of active site variants and mechanistic characterization of the aberrant self-hydroxylation chemistry. We continued to characterize active site variants of two bacterial herbicide-degrading enzymes, RdpA and SdpA, and unraveled the basis of their enantiospecificities. We devised a new assay procedure for representatives of this group of enzymes that will allow characterization of both the coupled and uncoupled reactions. We explored the ability of a trypanosomal protein to hydroxylate the methyl group in thymine within DNA, the first step in
formation of a modified base in this protozoan, but could detect no activity, and have cloned and begun to characterize the products of two putative thymine hydroxylases genes. We compared the properties of XanA, a fungal representative of this enzyme class that hydroxylates xanthine, as purified from Aspergillus nidulans and as the recombinant enzyme from E. coli. These studies were extended to identify alternate substrates and inhibitors of XanA, along with generation of a homology model. Finally, we purified and characterized a protein, AidB, whose regulation is coincident with AlkB, another member of this enzyme family. AidB was shown to be a DNA-binding flavoprotein that is suspected to function in DNA repair.
Impacts
The studies described here provide essential insight into the chemical mechanisms of this novel class of enzymes. Because related enzymes carry out a wide variety of agriculturally significant reactions, the mechanistic studies associated with this project are likely to have long range ramifications in controlling and modifying the properties of many other important enzymes.
Publications
- Rohankhedkar, M., Mulrooney, S.B., Wedemeyer, W.J., Hausinger, R.P. 2006. The AidB component of the Escherichia coli adaptive response to alkylating agents is a flavin-containing, DNA-binding protein. J. Bacteriol. 188:223-230.
- Koehntop, K.D., Marimanikkuppam, S., Ryle, M.J., Hausinger, R.P., Que, L. 2006. Self-hydroxylation of taurine/alpha-ketoglutarate dioxygenase. Evidence for more than one oxygen activation mechanism. J. Biol. Inorg. Chem. 11:63-72.
- Luo, L., Pappalardi, M.B., Tummino, P.J., Copeland, R.A., Fraser, M.E., Grzyska, P.K., Hausinger, R.P. 2006. An assay for Fe(II)/2-oxoglutarate-dependent dioxygenases by enzyme-coupled detection of succinate formation. Anal. Biochem. 353:69-74.
- Mueller, T.A., Zavodszky, M.I., Feig, M.,Kuhn, L.A., Hausinger, R.P. 2006. Structural basis for the enantiospecificities of R- and S-specific phenoxypropionate/alpha-ketoglutarate dioxygenases. Protein Sci. 15:1356-1368.
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Progress 01/01/05 to 12/31/05
Outputs
This project is focused on characterizing several ferrous ion- and alpha-ketoglutarate-dependent dioxygenases that hydroxylate a diverse array of primary substrates while decomposing the alpha-ketoacid to carbon dioxide plus succinate. Detailed mechanistic studies center on the Escherichia coli enzyme TauD, which hydroxylates taurine (2-aminoethanesulfonate) to form an unstable intermediate that spontaneously generates sulfite and aminoacetaldehyde. These studies include the construction of enzyme variants and their analysis by use of steady-state and rapid reaction kinetic approaches, including stopped-flow ultraviolet-visible spectroscopy and continuous flow resonance Raman spectroscopy. A novel intermediate in catalysis has been identified as an Fe(IV)-oxo species. Detailed characterization of heavy metal inhibition was carried out. A second class of enzymes being studied utilize phenoxyalkanoic acid herbicides (e.g., 2,4-D, mecoprop, dichlorprop) as their primary
substrates. Of particular interest here are the structural features of two enzymes that allow them to use opposite enantiomers of phenoxypropanoates. Homology modeling and substrate docking approaches were coupled to mutagenesis and kinetic studies to identify the basis for enantiospecificity. A third project involves the characterization of enzymes using DNA as substrate, in particular we are further characterizing the E. coli DNA repair enzyme AlkB by mutagenesis studies and examining a comparable enzyme in a moderate thermophile. A fourth subproject centers on a fungal enzyme that hydroxylates xanthine to uric acid using this chemistry, rather than the more traditional molybdenum-based mechanism. Finally, we are attempting to determine the role of the E. coli CsiD protein, which is a member of this enzyme according to its known crystal structure.
Impacts
The studies described here provide essential insight into the chemical mechanisms of this novel class of enzymes. Because related enzymes carry out a wide variety of agriculturally significant reactions, the mechanistic studies associated with this project are likely to have long range ramifications in controlling and modifying the properties of many other important enzymes.
Publications
- Grzyska, P.K., Ryle, M.J., Monterosso, G.R., Liu, J., Ballou, D.P., Hausinger, R.P. 2005. Steady-state and transient kinetic analyses of taurine/alpha-ketoglutarate dioxygenase: effects of oxygen concentration, alternative sulfonates, and active site variants on the Fe(IV)-oxo intermediate. Biochemistry 44:3845-3855.
- Kalliri, E., Grzyska, P.K., Hausinger, R.P. 2005. Kinetic and spectroscopic investigation of Co(II), Ni(II), and N-oxalylglycine inhibition of the Fe(II)/alpha-ketoglutarate dioxygenase, TauD. Biochem. Biophys. Res. Commun. 338:191-197.
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Progress 01/01/04 to 12/31/04
Outputs
This project focuses on characterization of several ferrous- and alpha-ketoglutarate-dependent dioxygenases that hydroxylate their primary substrates while decomposing alpha-ketoglutarate to carbon dioxide plus succinate. Two related herbicide-degrading enzymes from Sphingomonas herbicidovorans, SdpA and RdpA, metabolize the broad-leaf herbicides mecoprop and dichlorprop to form pyruvate and the modified phenols. Significantly, these enzymes utilize opposite enantiomers of the substrate, S- and R- enantiomers, respectively. In order to better understand the basis of the enantiospecificity, we have modeled the two enzyme structures on the basis of their similarity to structurally characterized family members. In addition, we have utilized site-directed mutagenesis to convert target residues at the proposed active sites in an effort to reverse the enantiospecificities. The taurine-degrading enzyme from Escherichia coli, TauD, decomposes 2-aminoethanesulfonate to
aminoacetaldehyde and sulfite. We have characterized a suite of enzyme variants for their steady-state kinetics and for the transient kinetics associated with a spectroscopically detected Fe(IV)-oxo intermediate. The active site of the enzyme also was probed by sophisticated electron paramagnetic resonance spectroscopy approaches for sample with bound nitric oxide, an oxygen analogue that converts the metal center to a paramagnetic site. The Escherichia coli DNA repair enzyme AlkB hydroxylates the methyl groups of 1-methyladenine or 3-methylcytosine in alkylated DNA or RNA to form unstable intermediates that decay to release formaldehyde and restore the undamaged base. We have carried out site-directed mutagenesis of this protein in order to better define its DNA-binding site and to better characterize its mechanism of catalysis.
Impacts
The studies described here provide essential insight into the chemical mechanisms of this novel class of enzymes. Because related enzymes carry out a wide variety of agriculturally significant reactions, the mechanistic studies associated with this project are likely to have long range ramifications in controlling and modifying the properties of many other important enzymes.
Publications
- Henshaw, T.F., Feig, M., Hausinger, R.P. 2004. Aberrant activity of the DNA repair enzyme AlkB. J. Inorg. Biochem. 98:856-861.
- Proshlyakov, D. A., Henshaw, T. F. , Monterosso, G. R., Ryle, M. J., Hausinger, R. P. 2004. Direct detection of oxygen intermediates in the non-heme Fe enzyme taurine/alpha-ketoglutarate dioxygenase. J. Am. Chem. Soc. 126:1022-1023.
- Hausinger, R. P. 2004. Fe(II)/alpha-ketoglutarate-dependent hydroxylases and related enzymes. Crit. Rev. Biochem. Molec. Biol. 39:21-68.
- Henshaw, T. F. 2004. Studies of two iron-containing enzymes from Escherichia coli: PFL-AE and AlkB. Ph.D. thesis.
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Progress 01/01/03 to 12/31/03
Outputs
This project focused on characterization of three ferrous ion- and alpha-ketoglutarate-dependent dioxygenases that hydroxylate their primary substrates while decomposing alpha-ketoglutarate to carbon dioxide plus succinate. The first, TfdA from Ralstonia eutropha, carries out the decomposition of the herbicide 2,4-dichlorophenoxyacetic acid to produce glyoxylate and 2,4-dichlorophenol. The protein was found to exhibit endogenous fluorescence from tryptophan residues, and the fluorescence quenching induced by the binding of ferrous ion and alpha-ketoglutarate was used to characterize the binding affinities of these compounds. Site directed mutagenesis methods were used to replace the tryptophan residues with alternate side chains, to identify the critical residues at the active site. The second enzyme of this family, TauD from Escherichia coli, hydroxylates taurine (2-aminoethanesulfonate) to form an unstable intermediate that decomposes to aminoacetaldehyde and
sulfite. This protein was shown by ultraviolet-visibel and electron paramagetic resonance spectroscopy to generate a reactive oxygen intermediate that abstracts a hydrogen atom from a tyrosyl side chain to transiently produce a tyrosyl radical that subsequently reacts with solvent to yield a catechole. The modified tyrosine was identified by site directed mutagenesis to be Tyr 73 in the protein. When formed by the decomposition of alpha-ketoglutarate, the catechol binds the metal to yield a spectral intermediate with maximum absorption at 550 nm, whereas when the catechol is formed by hydrogen peroxide treatment of protein with bound succinate the spectral maximum is located at 700 nm. These differing results were identified as arising from the same metallocenter species with either bicarbonate or solvent bound to the metal by the use of resonance Raman spectroscopy. Continuous flow resonance Raman methods were used to examine the reactive oxygen intermediate in the enzyme and
provided evidence consistent with the presence of an Fe(IV)-oxo species. The third enzyme of this family, AlkB from Escherichia coli, participates in DNA repair of alkylated DNA. In the absence of this substrate, the enzyme was shown to catalyze an aberrant side reaction in which a tryptophan side chain is hydroxylated. Structural prediction methods were used to show that the modified residue is located at the active site.
Impacts
The studies described here provide essential insight into the chemical mechanisms of this novel class of enzymes. Because related enzymes carry out a wide variety of agriculturally significant reactions, the mechanistic studies associated with this project are likely to have long range ramifications in controlling and modifying the properties of many other important enzymes.
Publications
- Dunning Hotopp, J.C., Auchtung, T.A., Hogan, D.A., and Hausinger, R.P. 2003. Intrinsic tryptophan fluorescence as a probe of metal and alpha-ketoglutarate binding to TfdA, a mononuclear non-heme iron oxygenase. J. Inorg. Biochem. 93:66-70.
- Ryle, M. J., Liu, A., Muthukumaran, R.B., Ho, R.Y.N., Koehntop, K.D., McCracken, J., Que, Jr., L., and Hausinger, R.P. 2003. O2- and alpha-ketoglutarate-dependent tyrosyl radical formation in TauD, an alpha-keto acid-dependent non-heme iron dioxygenase. Biochemistry 42:1854-1862.
- Ryle, M. J., Koehntop, K.D., Liu, A., Que, Jr., L., and Hausinger, R.P. 2003. Interconversion of two oxidized forms of taurine/alpha-ketoglutarate dioxygenase, a non-heme iron hydroxylase: evidence for bicarbonate binding. Proc. Natl. Acad. Sci. USA 100:3790-3795.
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Progress 01/01/02 to 12/31/02
Outputs
This project focused on characterization of three alpha-ketoglutarate dependent dioxygenases that hydroxylate their primary substrates while decomposing alpha-ketoglutarate to carbon dioxide and succinate. The first, TfdA from Ralstonia eutropha, carries out the first step in the decomposition of the herbicide 2,4-dichlorophenoxyacetic acid to produce glyoxylate and 2,4-dichlorophenol. The three-dimensional structure was modeled by comparison to related proteins that had been crystallographically characterized, putative active site residues were identified, and site-directed mutagenesis carried out. The properties of the mutant proteins verified the importance of the suspected key residues. An acetylenic analogue of the substrate, phenylpropiolic acid was shown to be a mechanism-based inactivator of TfdA. The site of modification was identified by a combination of mutagenesis and mass spectrometric approaches. Genes encoding TfdA-like proteins were identified in a
wide range of other soil bacteria. The second enzyme of this family, TauD from Escherichia coli, hydroxylates 2-aminoethanesulfonate (taurine) to form an unstable compound that decomposes to sulfite and aminoacetaldehyde. The crystal structure of the enzyme was determined and the self-hydroxylation reactivity of the enzyme was characterized in the absence of taurine, both with oxygen and with hydrogen peroxide. The enzyme was shown to form a transient tyrosyl radical that reacted further to generate a catechol according to evidence from electron paramagnetic resonance and resonance Raman spectroscopies. The third enzyme of this superfamily, AlkB from Escherichia coli, participates in DNA repair of alkylated DNA. This enzyme hydroxylates methyl groups at the 1 position of adenine or the 3 position of cytosine within DNA. The products are unstable and decompose to restore unmodified DNA and formaldehyde.
Impacts
The studies described here set the stage for future efforts to harness the unique chemistry of the alpha-ketoglutarate dependent dioxygenases for desired bioremediation or other biodegradative efforts. In addition, the AlkB studies highlight a new mechanism for DNA repair that may have more widely applicable ramifications.
Publications
- Dunning Hotopp, J. C., and Hausinger, R. P. 2002. Probing the 2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenase substrate binding site by site-directed mutagenesis and mechanism-based inactivation. Biochemistry 41:9787-9794.
- Trewick, S. C., Henshaw, T. F., Hausinger, R. P., Lindahl, T., and Sedgwick, B. 2002. Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage. Nature 419:174-178.
- Dunning Hotopp, J. C. 2002. Characterization of the substrate specificity of 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dependent dioxygenase. Ph.D. thesis.
- Ryle, M. J., and Hausinger, R. P. 2002. Non-heme iron oxygenases. Curr. Opin. Chem. Biol. 6:193-201.
- Elkins, J. M., Ryle, M. J., Clifton, I. J., Lloyd, J. S., Dunning Hotopp, J. C., Burzlaff, Baldwin, J. E., Hausinger, R. P. and Roach, P. L. 2002. X-ray crystal structure of Escherichia coli taurine/alpah-ketoglutarate dioxygenase complexed to ferrous iron and substrates. Biochemistry 41:5185-5192.
- Itoh, K., Kanda, R., Sumita, Y., Kim, H., Kamagata, Y., Suyama, K., Yamamoto, H., Hausinger, R. P., and Tiedje, J. M. 2002. tfdA-like genes in 2,4-D-degrading bacteria belonging to the Bradyrhizobium-Agromonas-Nitrobacter-Afipia cluster in alpha-Proteobacteria. Appl. Environ. Microbiol. 68:3449-3454.
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Progress 01/01/01 to 12/31/01
Outputs
This project focused on characterization of two alpha-ketoglutarate-dependent dioxygenases that hydroxylate their primary substrates while decomposing alpha-ketoglutarate to carbon dioxide and succinate. The first, TauD from Escherichia coli, hydroxylates 2-aminoethanesulfonate to form an unstable compound that decomposes to sulfite and aminoacetaldehyde. The second, TfdA from Ralstonia eutropha, carries out the first step in decomposition of the herbicide 2,4-dichlorophenoxyacetic acid to produce glyoxylate and 2,4-dichlorophenol. The structure of TauD was elucidated by crystallographic methods, and the structure of TfdA (30% identical to TauD in sequence) was modeled. TfdA was shown to catalyze a self-hydroxylation reaction at Trp113 (adjacent to a predict metal ligand residue) resulting in formation of a blue chromophore. TauD catalyzes several self-hydroxylation reactions including modification of three Trp residues (Trp128, Trp240, and Trp248) and formation of a
crosslinked histidyl-trihydroxyphenylalanine involving His255 and Tyr256. The latter reaction involves a tyrosyl radical intermediate that we speculate may also participate in catalysis. TfdA was shown to react with a variety of substituted cinnamic acids, perhaps lending insight into the evolutionary precursor of this enzyme. The alpha-ketoglutarate bound form of TauD was examined by resonance Raman spectroscopy, providing a useful spectroscopic probe for conversion of the 6-coordinate metal state to a 5-coordinate state upon substrate binding. These results suggest the creation of an oxygen binding site to allow increased reactivity when substrate is bound. Preliminary studies with the Escherichia coli protein AlkB suggests that this DNA repair enzyme also belongs to the alpha-ketoglutarate dependent dioxygenase superfamily.
Impacts
The studies described here set the stage for future efforts to harness the unique chemistry of the alpha-ketoglutarate dependent dioxygenases for desired bioremediation or other biodegradative efforts.
Publications
- Dunning Hotopp, J.A., Hausinger, R.P. 2001. Alternative substrates of 2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenase. J. Molec. Catalysis 15:155-162.
- Ho, R.Y.N., Mehn, M.P., Hegg, E.L., Liu, A., Ryle, M.J., Hausinger, R.P., Que, L.,Jr. 2001. Resonance Raman studies of the iron(II)-alpha-keto acid chromophore in model and enzyme complexes. J. Am. Chem. Soc. 123:5022-5029.
- Liu, A., Ho, R.Y.N., Que, L., Jr., Ryle, M.J., Phinney, B.S., Hausinger, R.P. 2001. Alternative reactivity of an alpha-ketoglutarate-dependent iron(II) oxygenase: enzyme self-hydroxylation. J. Am. Chem. Soc. 123:5126-5127.
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Progress 01/01/00 to 12/31/00
Outputs
This project focused on characterization of two alpha-ketoglutarate-dependent dioxygenases that hydroxylate their primary substrates while decomposing alpha-ketoglutarate to carbon dioxide and succinate. The first, TfdA from Ralstonia eutropha, carries out the first step in decomposition of the herbicide 2,4-D (2,4-dichlorophenoxyacetic acid) by hydroxylating the side chain. The unstable intermediate subsequently decomposes to glyoxylate and 2,4-dichlorophenol. The second enzyme, TauD from Escherichia coli, hydroxylates 2-aminoethane sulfonate (taurine) to form an unstable compound that decomposes to sulfite and aminoacetaldehyde. We carried out site directed mutagenesis of TfdA accompanied by electron paramagnetic resonance and electron spin echo envelop modulation spectroscopies of the modified proteins in order to identify the critical residues at the metal-containing active site. We established that His113, Asp115, and His262 are the likely Fe(II) ligands, and
that His213 may be near the active site. Preliminary crystallographic studies of TauD (about 30% identical in sequence to TfdA) are consistent with these assignments. We showed that both TfdA and TauD catalyze self-hydroxylation reactions when the primary substrate is not available. The current data suggest that hydroxy-tryptophan is made in both proteins and trihydroxyphenylalanine is formed in TauD based on mass spectrometry, resonance Raman spectroscopy, fluorescence studies, and other work. The kinetics of these processes were examined and compared to the kinetics involving intermediates observed during the catalytic reaction.
Impacts
The results of these studies have wide relevance to the growing list of alpha-ketoglutarate-dependent dioxygenases. Our enzymes may serve as model systems to establish the underlying chemistry and mechanisms of these reactions. It is possible that new catalysts will be able to be generated by mutagenesis of these proteins to expand or alter their substrate range. This would offer the possibility of bioremediation of contaminated sites.
Publications
- Hogan, D. A., Smith, S. R., Saari, E. A., McCracken, J., and Hausinger, R. P. 2000. Site-directed mutagenesis of 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase. Identification of residues involved in metallocenter formation and substrate binding. J. Biol. Chem. 275:12400-12409.
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Progress 01/01/99 to 12/31/99
Outputs
The long-term goal of this project is to characterize the enzymatic mechanism of two iron- and alphaketoglutarate-dependent dioxygenases. The first enzyme, TfdA, carries out the first step in decomposition of the herbicide 2,4-D and may have an environmental role in metabolizing cinnamic acid-type compounds. The second enzyme, TauD, and its close relatives decompose taurine and other sulfonates to release sulfite and the corresponding aldehyde. We have analyzed the iron and copper-containing forms of TfdA by using a variety of biophysical spectroscopic approaches, and have demonstrated that the metal is bound by two histidine residues, that alphaketoglutarate binds to the metal, and that binding of 2,4-D alters the metal coordination. In addition, we characterized the stereochemistry of the oxidative reaction and found that different enzymes related in sequence can carry out opposite chemistry. Using TauD, we used stopped-flow electronic spectroscopy to establish the
kinetics of substrate binding and intermediate transformations.
Impacts
The results of these studies have wide relevance to the growing list of alphaketoglutarate-dependent dioxygenases, and our enzymes serve as model systems to establish the underlying chemistries of these reactions. In addition, these enzymes may have future biotechnological utility in bioremediation of herbicide or sulfonate-contaminated sites.
Publications
- Cosper, N.J., Stalhandske, C.V.M., Saari, R.E., Hausinger, R.P., Scott, R.A. 1999. X-ray absorption spectroscopic analysis of Fe(II) and Cu(II) forms of an herbicide-degrading alpha-ketoglutarate dioxygenase. J. Biol. Inorg. Chem. 4:122-129.
- Saari, R.E., Hogan, D.A., Hausinger, R.P. 1999. Stereospecific degradation of the phenoxypropionate herbicide dichlorprop. J. Molec. Catalysis B 6:421-428.
- Hogan, D.A., Auchtung, T.A., Hausinger, R.P. 1999. Cloning and characterization of a sulfonate/alpha-ketoglutarate dioxygenase from Saccharomyces cerevisiae. J. Bacteriol. 181:5876-5879.
- Ryle, M.J., Padmakumar, R., Hausinger, R.P. 1999. Stopped-flow kinetic analysis of Escherichia coli taurine/alpha-ketoglutarate dioxygenase: interactions with alpha-ketoglutarate, taurine, and oxygen. Biochemistry 38:15278-15286.
- Hegg, E.L., Whiting, A.K., Saari, R.E., McCracken, J., Hausinger, R.P., Que, L. 1999. The herbicide-degrading alpha-ketoacid-dependent enzyme TfdA: metal coordination environment and mechanistic insights. Biochemistry 38:16714-16726.
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