1986 — 1988 |
Hausinger, Robert P |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Characterization of Bacterial Ureases @ Michigan State University
The ultimate objective of this research is to provide a dtailed mechanistic understanding of bacterial urease, a nickel-containing enzyme. Elucidation of the urease mechanism potentially could lead to the develepment of specific enzyme inhibitors. Such inhibitors may have utility in several areas of human health, animal husbandry, and agronomy. For example, urinary stones which are induced by ureolytic bacteria infecting the urinary tract may be eliminated through the use of urease inhibitors; thus, the incidence of hospitalization for this conditon (1 per 1000 adults per year) may be significantly reduced. The experiments in this proposal work toward the goal of elucidating unrease action by focusing on the following specific aims. Urease will be isolated and chaacterized from Proteus mirabilis, the bacterium most often associated with urinary stone formation, Selenomonas ruminantium, an important ureolytic rumen microbe, Sporosarcina ureae, a soil bacterium which possesses high levels of urease, and Klebsiella aerogenes, a ureolytic bacterium which can be easily manipulated genetically. The urease from each organism will be analyzed for stability, native and subunit molecular weight, number of nickel per subunit, amino acid composition, amino terminal sequence, and kinetic parameters. The nickel-containing urease which is most readily purified in large quantities and has the largest potential for experimental flexibility will be chosen for in-depth studies. The nickel-site will be extracted and characterized, the nickel ligands will be identified, and reconstitution experiments will be performed. Potential inhibitors and inactivators of urease will be screened, and covalent inactivators will be used to identify active-site amino acid residues.
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1 |
1990 — 1992 |
Hausinger, Robert |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Mechanism of Nickel Incorporation Into Urease @ Michigan State University
The biosynthesis of enzyme metallocenters is very poorly understand. This proposal is aimed at understanding the general mechanism for incorporation of the metal into the protein matrix using urease as a model. Urease, synthesized as an apo-enzyme, requires an accessory factor for the formation of its novel, bi- nickel active site. The nature of the accessory factor is unknown; however, two newly identified genes appear to play a role in this process. Mutants of these genes will be generated and the effects of the mutations on the apo-protein will be determined. The accessory protein (s) will be isolated and characterized for cellular location, Ni binding and interaction with apo-urease. The research will increase our understanding of how metalloproteins are formed, and our understanding of the biochemistry of nickel in living systems.
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0.915 |
1994 — 2010 |
Hausinger, Robert P |
P41Activity Code Description: Undocumented code - click on the grant title for more information. R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanism of Urease Metallocenter Biosynthesis @ Michigan State University
DESCRIPTION (provided by applicant): The studies described in this proposal focus on the mechanism of activation of bacterial urease, a bacterial virulence factor. The urease apoprotein (UreABC) serves as the scaffold for creation of a novel lysine carbamate-bridged, dinuclear nickel active site metallocenter. Our working model of the assembly process requires the action of a GTP-dependent molecular chaperone (made up of UreD, UreF, and UreG accessory proteins) along with the participation of a metallochaperone (UreE) that delivers Ni. We seek to understand the structure and function of each urease accessory protein and to elucidate the mechanism by which these proteins participate in this unique GTP-dependent process of metallocenter assembly. Our objectives are to test specific hypotheses regarding the roles of the urease accessory proteins. Thus, we will (A) Carry out structural analyses of the individual UreD, UreF, and UreG components along with the UreDFG heterotrimer. (B) Unravel the sequential binding kinetics associated with formation of the UreD-UreABC, UreDF-UreABC, and UreDFG-UreABC species. (C) Establish the overall conformation of these complexes by small angle X-ray scattering methods. (D) Identify the specific sites of protein:protein interaction by developing an innovative "protein footprinting" technology that should be widely applicable to other systems. (E) Explore the timing and stability of lysine carbamylation within these complexes by comparing the incorporation of radiolabeled CO2 with and without trapping by diazomethane. (F) Seek to stabilize the putative UreE:UreG interaction by use of mutants and exchange-inert metal ions. (G) Test for the creation of a high affinity Ni-binding site at the UreE:UreG interface by using isothermal titration calorimetry and other metal-binding approaches. (H) Examine the effect of MgGTP and its analogues on conformational changes associated with metal transfer into UreABC. The general processes involved in urease metallocenter biosynthesis are likely to apply to numerous other systems, including the activation of many metalloenzymes of medical interest. Significantly, our focus on understanding urease activation will uncover common principles that can be applied to these less tractable systems. In addition, we will expand our knowledge regarding the biochemistry of nickel.
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1 |
1997 |
Hausinger, Robert P |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Bacterial Cell Mechanism of Urease Nickel Ion Biosynthesis @ Michigan State University
technology /technique development; proteins; enzymes; biomedical resource; biological products;
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1 |
1997 |
Hausinger, Robert P |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Overcoming Barriers to in Situ Biological Remediation of Pcbs @ Michigan State University
environmental toxicology; plants; environmental health; biomedical resource; bioengineering /biomedical engineering; biological products; bacteria;
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1 |
2002 — 2005 |
Hausinger, Robert P |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Study of Alpha-Ketoglutarate-Dependent Dioxygenases @ Michigan State University
DESCRIPTION (provided by applicant): Enzymes containing mononuclear non-heme iron sites catalyze a diverse array of reactions that are significant to medicine and to the environment. This proposal describes plans to study three representatives of the largest, but perhaps least well understood, grouping of these enzymes: The a-ketoglutarate (aKG)-dependent dioxygenase superfamily. TauD catalyzes the hydroxylation of taunne and other sulfonates as a first step in their metabolism. TfdA carries out similar chemistry during catabolism of the herbicide 2,4-dichiorophenoxyacetic acid (2,4-D). Phytanoyl-CoA hydroxylase, a new research direction for this laboratory, is required for utilization of C-3 branched fatty acids; deficiencies of this human enzyme lead to Refsum disease, rhizomelic chondrodysplasia punctata, and ZeHweger syndrome. The specific aims include: (1) Characterize the enzyme mechanism of TauD and TfdA by examining the properties of catalytic intermediates and analyzing the effects of site-directed mutagenesis. (2) Examine the biogenesis of the tyrosyl radical, hydroxy-tryptophan, and histidyl-trihydroxyphenylalanine found in TauD and identify the structures & synthesis of modifications present in TfdA. (3) Obtain high-resolution three-dimensional protein structures of TauD, TfdA, and their variants in their various states. (4) Explore the metallocenter properties of phytanoyl-CoA hydroxylase using the recombinant human enzyme and/or a. more tractable microbial model system. Of particular interest will be studies to test a new mechanism for this enzyme superfamily. Specifically, we propose that these enzymes possess catalytically essential tyrosine residues that are used to resonance stabilize Fe(IV)=O as Fe(III)-OH/tyrosine radical states.
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2006 — 2009 |
Hausinger, Robert P |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Characterization Fo Fe(Ii)Alpha-Ketoglutarate-Dependent Hydroxylases @ Michigan State University
[unreadable] DESCRIPTION (provided by applicant): Enzymes containing mononuclear non-heme iron sites catalyze a diverse array of reactions that are significant to medicine and to the environment. The studies described in this proposal focus on the largest group of non-heme iron enzymes, the Fe(ll)- and alpha-ketoglutarate (aKG)-dependent hydroxylases. We seek to better define the hydroxylase reaction intermediates, enhance our understanding of the protein features involved in substrate recognition, and expand our knowledge of the functional roles of related family members. The specific aims include: (1) Use variants of the best-studied representative of this enzyme family, the sulfonate-metabolizing enzyme TauD, to spectroscopically analyze the Fe(IV)-oxo intermediate, examine other catalytic species, and to determine mutant protein structures. Also, investigate TauD interactions with inhibitors and study quantitatively the self-hydroxylation chemistry of this enzyme to test hypotheses regarding the role of the enzyme side chain modification reactions. (2) Elucidate the interactions of the E. coli DMA-repair enzyme AlkB with its substrate, methylated DNA, and investigate the roles of several human homologues. (3) Structurally and spectroscopically characterize the herbicide-degrading enzyme TfdA, and determine the basis for the opposite enahtiospecificities of two related enzymes, RdpA and SdpA, by using structural and mutagenesis approaches. (4) Define the structure, biochemical properties, and spectroscopically accessible catalytic intermediates of a newly identified Fe(ll)/aKG-dependent hydroxylase that oxidizes xanthine. (5) Identify the function of the E. coli Gab protein and spectroscopically examine its catalytic intermediates. (6) Explore the potential for carefully selected Fe(ll)/aKG hydroxylase family members to function in criromatin demethylation. The first three aims continue ongoing investigations in the laboratory, while the latter three aims present new research directions. Insights gained from these studies will be useful in understanding the substrate recognition features and chemical mechanisms of a large number of enzymes, including many less tractable examples that have direct medical relevance. [unreadable] [unreadable] [unreadable]
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2011 — 2014 |
Hausinger, Robert P |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Characterization of Fe(Ii)/Alpha-Ketoglutarate-Dependent Hydroxylases @ Michigan State University
DESCRIPTION (provided by applicant): This project examines the enzymatic mechanism used by FeII/?-ketoglutarate (?KG)-dependent hydroxylases and explores the diversity of reactions they catalyze. Members of this enzyme family are widespread in bacteria and eukaryotes (including humans) where they promote reactions of fundamental importance including DNA/RNA repair, synthesis/degradation of a vast repertoire of small molecules, lipid metabolism, and protein hydroxylation related to oxygen sensing, chromatin demethylation, or structural interactions. The studies detailed in this proposal focus on four aims. First, we will define the chemical steps during early catalysis by applying an innovative continuous- flow Raman spectroscopic approach to TauD, the best studied member of this enzyme family. Of special interest will be the properties of a key TauD variant that slowly forms the known FeIV=O intermediate, as well as the behavior of a thermophilic homologue. Parallel studies will probe for uniformity of the identified reaction intermediates in two other available family members. Second, pulsed EPR techniques will be utilized to investigate the geometries of active site environments for enzymes with bound nitric oxide (NO), a surrogate of O2. Measurements using these novel methods will be validated with TauD, where we have crystallographic information, and then applied to XanA, a xanthine-degrading enzyme, for which structural data are lacking. In particular, these techniques will be exploited to probe small structural changes at the active site upon substrate binding or in selected variant proteins. Third, the presence of a second FeII binding site in TauD will be confirmed and the function of this site will be investigated. As part of these studies, we will explore the use of phosphorescence quenching to obtain thermodynamic binding data on anaerobic proteins. Finally, biochemical and spectroscopic properties will be elucidated for TET1, a 5-meC hydroxylase that might function with another enzyme as a DNA demethylase. In total, this work will enhance our understanding of the enzyme mechanism common to this versatile enzyme family while further defining new and diverse roles for its individual members. Such studies have medical relevance because understanding of this mechanism is critical for developing treatments of human genetic diseases associated with defects in FeII/?KG hydroxylases, for defending against pathogens where such enzymes play essential roles, and for optimizing the synthesis of antibiotics by these enzymes in other microbes.
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2015 — 2018 |
Hu, Jian (co-PI) [⬀] Hausinger, Robert |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Structure, Mechanism and Nickel Metallocenter Assembly of Lactate Racemase @ Michigan State University
With this award, the Chemistry of Life Processes Program in the Division of Chemistry is funding Professors Robert P. Hausinger and Jian Hu of Michigan State University to characterize the structures and functions of enzymes involved in lactate biosynthesis. The enzyme that is the focus of this study, lactate racemase, interconverts two forms of lactic acid, in a deceptively simple, though chemically challenging, reaction. Information gained with these complexes may be used to inform other, studies of enzymes dependent upon nickel metal centers. This research trains postdoctoral researchers in the use of mass spectrometric, structural, spectroscopic, and mutagenesis studies to characterize the enzyme and its helper proteins. These individuals interact with gifted high school students in the High School Honors Science Program and underrepresented undergraduate students in the Charles Drew Science Scholars program, the Increasing Diversity and Education Access to Sciences (IDEAS) program, and the Summer Research Opportunities Program (SROP). Furthermore, information gained from this project is incorporated into ongoing graduate courses entitled "Metals in Biology," "Protein Structure, Function, and Design," and "Integrated Microbial Biology."
This project examines the functional role and biosynthetic pathway of a recently identified pyridinium-3-thioamide-5-thiocarboxylic acid mononucleotide that coordinates nickel as a (SCS)Ni(II) pincer complex and is covalently bound to Lys184 of lactate racemase, LarA. Synthesis of this cofactor requires the accessory proteins LarB, LarC, and LarE of still undefined roles. Planned analyses include investigation of protein bound and free intermediates by mass spectrometry, structural elucidation of these proteins in their various states by x-ray crystallography, spectrophotometric and biophysical analyses of LarA intermediates during catalysis, and in vitro recapitulation of the biosynthetic pathway by supplying the appropriate cofactors. This research is expected to expand understanding of nickel biochemistry and elucidate mechanistic details of oxidation-reduction chemistry involving a tethered, nickel-containing, nicotinic acid cofactor. The (SCS)Ni(II) cofactor represents the first biological example of a pincer complex, well known to inorganic chemists.
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0.915 |
2019 — 2025 |
Hausinger, Robert Hu, Jian (co-PI) [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Collaborative Research: Ethylene-Forming Enzyme @ Michigan State University
Guanidine is a potential nitrogen fertilizer and ethylene has been proposed as a potential alternative fuel source; thus, the biosynthetic reactions that form these compounds are commercially important as they have potential benefits for agriculture and the biofuels industries. With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Drs. Robert Hausinger and Jian Hu at Michigan State University, and Dr. Christo Christov at Michigan Technological Institute to investigate how ethylene-forming enzyme (EFE) from bacteria and fungi catalyzes dual reactions to form guanidine or ethylene. In the typical reaction of EFE, a carbon dioxide is removed from other molecules (2-oxoglutarate, or 2OG), while being added to the amino acid, L-arginine, to transform it into guanidine. Alternatively, EFE catalyzes the transformation of 2OG into ethylene, with the release of carbon dioxide / bicarbonate. Research into the mechanisms of EFE catalysis provides specialized biochemical, structural, and computational training to postdoctoral fellows, and graduate and undergraduate students, including women and students from underrepresented minority groups. Incorporation of these studies into a university science festival engages families and lifelong learners through outreach programs about the potential use of ethylene as a biofuel.
The overarching goals of this research project are to elucidate the molecular determinants that distinguish the two distinct reactions catalyzed by EFE and to establish the catalytic mechanisms of the enzyme by a combination of experimental and computational studies. This work also investigates why other members of the iron(II)- and 2OG-dependent oxygenases fail to produce ethylene. To understand the basis of the two EFE-catalyzed reactions, the researchers are characterizing naturally occurring analogs, a reconstructed ancestor, and site-directed variants of the protein by using kinetic, structural, and computational methods. Using this information along with structure-guided protein engineering, the researchers are creating EFE variants optimized for the production of either the biofuel ethylene or the plant fertilizer guanidine while eliminating wasteful L-Arginine hydroxylation or 2OG-degrading reactions. In addition, the team is using biochemical, kinetic, spectroscopic, and computational methods to elucidate the chemical reaction mechanism of EFE. This work extends the structural/mechanistic information available on other important iron(II)- and 2OG-dependent oxygenases that are unable to catalyze these reactions.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
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0.915 |
2019 — 2021 |
Hausinger, Robert P Hu, Jian (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Nickel-Pincer Nucleotide Enzymes @ Michigan State University
Project Summary/Abstract The nickel-pincer nucleotide (NPN) cofactor is a newly identified coenzyme discovered in lactate racemase (LarA) from Lactobacillus plantarum. Synthesis of the active enzyme requires the participation of three accessory proteins that act in sequence: LarB carboxylates the pyridinium ring and hydrolyzes the phosphoanhydride of nicotinic acid adenine dinucleotide, LarE converts the two pyridinium ring carboxylates to thiocarboxylates, and LarC inserts nickel (forming two S-Ni and one C-Ni bonds) during synthesis of the novel cofactor. Genes encoding these four proteins are widely distributed in microorganisms associated with the human microbiome and among human pathogens. The long-term objective of the effort described here is to advance significantly our understanding of how microorganisms, including pathogenic species, make and utilize the NPN cofactor. Two specific aims will achieve this objective: (1) characterize the components of the NPN biosynthetic systems and (2) identify the roles of the NPN cofactor in lactate racemase and additional enzymes. Investigations of LarB will define the structure and mechanism of this pyridinium ring carboxylase/phosphoanhydride hydrolase. Studies of a multi-cysteine and probable [4Fe4S]-containing form of LarE will establish whether it operates by a catalytic sulfur-transfer mechanism, in contrast to the sacrificial LarE of L. plantarum with its single active site cysteine that converts to dehydroalanine. Structural and mechanistic analysis of the CTP-dependent nickel-inserting LarC will elucidate how this protein installs nickel into the cofactor. The geometry of lactate binding to L. plantarum LarA will be defined, the full range of substrates used by this enzyme will be established, and substrates will be identified for alternative LarA-like proteins. Proteins that covalently bind the NPN cofactor will be identified and characterized using innovative chemistry that reacts the coenzyme with a fluorescent tag. Radioactive nickel (63Ni) and 14C-nicotinic acid also will be used to label new NPN cofactor-binding proteins, followed by mass spectrometry, bioinformatics, and biochemical studies to identify the functions of these novel enzymes. The findings obtained through these efforts will greatly increase knowledge of the synthesis and utilization of nickel- pincer cofactors in bacteria, including those important to human health, with implications for identification of potential antimicrobial drug targets.
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