Chemical Rescue and Inhibition Studies to Determine the Role of Arg301 in Phosphite Dehydrogenase
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{"title"=>"Chemical rescue and inhibition studies to determine the role of Arg301 in phosphite dehydrogenase", "type"=>"journal", "authors"=>[{"first_name"=>"John E.", "last_name"=>"Hung", "scopus_author_id"=>"55233879800"}, {"first_name"=>"Emily J.", "last_name"=>"Fogle", "scopus_author_id"=>"10142568100"}, {"first_name"=>"Neha", "last_name"=>"Garg", "scopus_author_id"=>"35368627000"}, {"first_name"=>"Jonathan R.", "last_name"=>"Chekan", "scopus_author_id"=>"55966594400"}, {"first_name"=>"Satish K.", "last_name"=>"Nair", "scopus_author_id"=>"7402726311"}, {"first_name"=>"Wilfred A.", "last_name"=>"Van Der Donk", "scopus_author_id"=>"7003819176"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"isbn"=>"1932-6203", "pmid"=>"24498026", "doi"=>"10.1371/journal.pone.0087134", "pui"=>"373059491", "issn"=>"19326203", "sgr"=>"84900299073", "scopus"=>"2-s2.0-84900299073"}, "id"=>"54a14784-2a65-3f7f-a89b-b7dfce76e96e", "abstract"=>"Phosphite dehydrogenase (PTDH) catalyzes the NAD(+)-dependent oxidation of phosphite to phosphate. This reaction requires the deprotonation of a water nucleophile for attack on phosphite. A crystal structure was recently solved that identified Arg301 as a potential base given its proximity and orientation to the substrates and a water molecule within the active site. Mutants of this residue showed its importance for efficient catalysis, with about a 100-fold loss in k cat and substantially increased K m,phosphite for the Ala mutant (R301A). The 2.35 Å resolution crystal structure of the R301A mutant with NAD(+) bound shows that removal of the guanidine group renders the active site solvent exposed, suggesting the possibility of chemical rescue of activity. We show that the catalytic activity of this mutant is restored to near wild-type levels by the addition of exogenous guanidinium analogues; Brønsted analysis of the rates of chemical rescue suggests that protonation of the rescue reagent is complete in the transition state of the rate-limiting step. Kinetic isotope effects on the reaction in the presence of rescue agents show that hydride transfer remains at least partially rate-limiting, and inhibition experiments show that K i of sulfite with R301A is ∼400-fold increased compared to the parent enzyme, similar to the increase in K m for phosphite in this mutant. The results of our experiments indicate that Arg301 plays an important role in phosphite binding as well as catalysis, but that it is not likely to act as an active site base.", "link"=>"http://www.mendeley.com/research/chemical-rescue-inhibition-studies-determine-role-arg301-phosphite-dehydrogenase", "reader_count"=>12, "reader_count_by_academic_status"=>{"Unspecified"=>3, "Researcher"=>4, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>3, "Student > Postgraduate"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>3, "Researcher"=>4, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>3, "Student > Postgraduate"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>3, "Biochemistry, Genetics and Molecular Biology"=>2, "Agricultural and Biological Sciences"=>5, "Chemical Engineering"=>1, "Chemistry"=>1}, "reader_count_by_subdiscipline"=>{"Chemistry"=>{"Chemistry"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>5}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>2}, "Unspecified"=>{"Unspecified"=>3}, "Chemical Engineering"=>{"Chemical Engineering"=>1}}, "reader_count_by_country"=>{"United States"=>1, "Japan"=>1}, "group_count"=>1}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1371322", "https://ndownloader.figshare.com/files/1371323", "https://ndownloader.figshare.com/files/1371324", "https://ndownloader.figshare.com/files/1371325", "https://ndownloader.figshare.com/files/1371326", "https://ndownloader.figshare.com/files/1371327", "https://ndownloader.figshare.com/files/1371328", "https://ndownloader.figshare.com/files/1371329", "https://ndownloader.figshare.com/files/1371330"], "description"=>"<div><p>Phosphite dehydrogenase (PTDH) catalyzes the NAD<sup>+</sup>-dependent oxidation of phosphite to phosphate. This reaction requires the deprotonation of a water nucleophile for attack on phosphite. A crystal structure was recently solved that identified Arg301 as a potential base given its proximity and orientation to the substrates and a water molecule within the active site. Mutants of this residue showed its importance for efficient catalysis, with about a 100-fold loss in <i>k</i><sub>cat</sub> and substantially increased <i>K</i><sub>m,phosphite</sub> for the Ala mutant (R301A). The 2.35 Å resolution crystal structure of the R301A mutant with NAD<sup>+</sup> bound shows that removal of the guanidine group renders the active site solvent exposed, suggesting the possibility of chemical rescue of activity. We show that the catalytic activity of this mutant is restored to near wild-type levels by the addition of exogenous guanidinium analogues; Brønsted analysis of the rates of chemical rescue suggests that protonation of the rescue reagent is complete in the transition state of the rate-limiting step. Kinetic isotope effects on the reaction in the presence of rescue agents show that hydride transfer remains at least partially rate-limiting, and inhibition experiments show that <i>K</i><sub>i</sub> of sulfite with R301A is ∼400-fold increased compared to the parent enzyme, similar to the increase in <i>K</i><sub>m</sub> for phosphite in this mutant. The results of our experiments indicate that Arg301 plays an important role in phosphite binding as well as catalysis, but that it is not likely to act as an active site base.</p></div>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Protein chemistry", "Protein interactions", "biocatalysis", "chemical biology", "enzymes", "biotechnology", "proteomics", "Chemical reactions", "catalysis", "inhibition", "studies", "arg301", "Phosphite"], "article_id"=>922170, "categories"=>["Biological Sciences", "Chemistry"], "users"=>["John E. Hung", "Emily J. Fogle", "Neha Garg", "Jonathan R. Chekan", "Satish K. Nair", "Wilfred A. van der Donk"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0087134.s001", "https://dx.doi.org/10.1371/journal.pone.0087134.s002", "https://dx.doi.org/10.1371/journal.pone.0087134.s003", "https://dx.doi.org/10.1371/journal.pone.0087134.s004", "https://dx.doi.org/10.1371/journal.pone.0087134.s005", "https://dx.doi.org/10.1371/journal.pone.0087134.s006", "https://dx.doi.org/10.1371/journal.pone.0087134.s007", "https://dx.doi.org/10.1371/journal.pone.0087134.s008", "https://dx.doi.org/10.1371/journal.pone.0087134.s009"], "stats"=>{"downloads"=>24, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Chemical_Rescue_and_Inhibition_Studies_to_Determine_the_Role_of_Arg301_in_Phosphite_Dehydrogenase_/922170", "title"=>"Chemical Rescue and Inhibition Studies to Determine the Role of Arg301 in Phosphite Dehydrogenase", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2014-01-31 03:16:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/1371317"], "description"=>"<p>The errors given in parentheses were obtained from fits of the experimental data to the appropriate equations. na = not applicable. nd = not determined. <i>k</i><sub>rescue</sub> refers to <i>k</i><sub>cat</sub> of the enzyme in cases where no rescue reagent was added (i.e. entries 1–3). <sup>a</sup>Data from reference <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087134#pone.0087134-Perrin1\" target=\"_blank\">[48]</a>.<sup> b</sup>Data from reference <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087134#pone.0087134-Hung1\" target=\"_blank\">[13]</a>.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Protein chemistry", "Protein interactions", "biocatalysis", "chemical biology", "enzymes", "biotechnology", "proteomics", "Chemical reactions", "catalysis", "constants"], "article_id"=>922165, "categories"=>["Biological Sciences", "Chemistry"], "users"=>["John E. Hung", "Emily J. Fogle", "Neha Garg", "Jonathan R. Chekan", "Satish K. Nair", "Wilfred A. van der Donk"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0087134.t001", "stats"=>{"downloads"=>5, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Steady_state_constants_for_chemical_rescue_of_activity_on_R301A_/922165", "title"=>"Steady state constants for chemical rescue of activity on R301A.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-01-31 03:16:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/1371316"], "description"=>"_", "links"=>[], "tags"=>["Biochemistry", "proteins", "Protein chemistry", "Protein interactions", "biocatalysis", "chemical biology", "enzymes", "biotechnology", "proteomics", "Chemical reactions", "catalysis", "inhibition", "arg301", "mutants"], "article_id"=>922164, "categories"=>["Biological Sciences", "Chemistry"], "users"=>["John E. Hung", "Emily J. Fogle", "Neha Garg", "Jonathan R. Chekan", "Satish K. Nair", "Wilfred A. van der Donk"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0087134.t002", "stats"=>{"downloads"=>6, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Sulfite_inhibition_in_Arg301_mutants_of_17X_PTDH_/922164", "title"=>"Sulfite inhibition in Arg301 mutants of 17X-PTDH.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-01-31 03:16:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/1371312"], "description"=>"<p>The active site of the thermostable PTDH in complex with NAD<sup>+</sup> and the competitive inhibitor sulfite <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087134#pone.0087134-Zou1\" target=\"_blank\">[17]</a>, depicting the potential active site bases His292 and Arg301.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Protein chemistry", "Protein interactions", "biocatalysis", "chemical biology", "enzymes", "biotechnology", "proteomics", "Chemical reactions", "catalysis", "ternary"], "article_id"=>922160, "categories"=>["Biological Sciences", "Chemistry"], "users"=>["John E. Hung", "Emily J. Fogle", "Neha Garg", "Jonathan R. Chekan", "Satish K. Nair", "Wilfred A. van der Donk"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0087134.g001", "stats"=>{"downloads"=>0, "page_views"=>18, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Depiction_of_the_active_site_PTDH_NAD_Sulfite_ternary_complex_/922160", "title"=>"Depiction of the active site PTDH-NAD<sup>+</sup>-Sulfite ternary complex.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-31 03:16:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/1371315"], "description"=>"<p>(A) Brønsted plot depicting the observed rates of phosphite oxidation by R301A in the presence of various rescue reagents at pH 7.25 as a function of their p<i>K</i><sub>a</sub>. β = 1.03±0.09 (r<sup>2</sup> = 0.96). (B) Dependence of the rate of chemical rescue on the concentration of aminoguanidine at pH 7.0 (black squares), pH 7.5 (red circles), and pH 8.0 (blue triangles). All data was corrected for the pH-dependence of <i>k</i><sub>cat,R301A</sub> (see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087134#pone.0087134.s004\" target=\"_blank\">Figure S4</a>). Also shown is the expected activity at pH 7.5 (red dashed line) and pH 8.0 (blue dashed line) based on the observed activity at pH 7.0 if the active reagent were the unprotonated aminoguanidine. Experiments were performed in duplicate; when error bars are not visible, the error was smaller than the marker.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Protein chemistry", "Protein interactions", "biocatalysis", "chemical biology", "enzymes", "biotechnology", "proteomics", "Chemical reactions", "catalysis"], "article_id"=>922163, "categories"=>["Biological Sciences", "Chemistry"], "users"=>["John E. Hung", "Emily J. Fogle", "Neha Garg", "Jonathan R. Chekan", "Satish K. Nair", "Wilfred A. van der Donk"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0087134.g003", "stats"=>{"downloads"=>2, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Chemical_rescue_plots_for_R301A_PTDH_/922163", "title"=>"Chemical rescue plots for R301A-PTDH.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-31 03:16:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/1371314"], "description"=>"<p>(A) Overlay of the crystal structures of 17X-PTDH (green) and the R301A mutant (blue) with NAD<sup>+</sup> bound. Residue 301 is shown with the immediately adjacent residues Trp134, Tyr139, and Ser295, which hydrogen bond to Arg301 in the 17X-PTDH structure. Also shown is the essential residue His292. (B) Surface model of the R301A mutant (grey) overlaid with Arg301 from 17X-PTDH (green) and Ala301 from R301A (blue) in sticks, showing the presence of a solvent-accessible channel into the substrate-binding pocket. For an overlay of 17X-PTDH with PTDH-R301K, see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087134#pone.0087134.s001\" target=\"_blank\">Figure S1</a>.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Protein chemistry", "Protein interactions", "biocatalysis", "chemical biology", "enzymes", "biotechnology", "proteomics", "Chemical reactions", "catalysis", "r301a"], "article_id"=>922162, "categories"=>["Biological Sciences", "Chemistry"], "users"=>["John E. Hung", "Emily J. Fogle", "Neha Garg", "Jonathan R. Chekan", "Satish K. Nair", "Wilfred A. van der Donk"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0087134.g002", "stats"=>{"downloads"=>1, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_X_ray_crystal_structure_of_the_R301A_mutant_/922162", "title"=>"X-ray crystal structure of the R301A mutant.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-31 03:16:39"}

PMC Usage Stats | Further Information

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Relative Metric

{"start_date"=>"2014-01-01T00:00:00Z", "end_date"=>"2014-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences", "average_usage"=>[291]}, {"subject_area"=>"/Biology and life sciences/Biochemistry", "average_usage"=>[282]}, {"subject_area"=>"/Biology and life sciences/Biotechnology", "average_usage"=>[310, 499]}, {"subject_area"=>"/Physical sciences/Physics", "average_usage"=>[266]}]}
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