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Title: N5-CAIR Mutase: Role of a CO2 Binding Site and Substrate Movement in Catalysis

Abstract

N{sup 5}-Carboxyaminoimidazole ribonucleotide mutase (N{sup 5}-CAIR mutase or PurE) from Escherichia coli catalyzes the reversible interconversion of N{sup 5}-CAIR to carboxyaminoimidazole ribonucleotide (CAIR) with direct CO{sub 2} transfer. Site-directed mutagenesis, a pH-rate profile, DFT calculations, and X-ray crystallography together provide new insight into the mechanism of this unusual transformation. These studies suggest that a conserved, protonated histidine (His45) plays an essential role in catalysis. The importance of proton transfers is supported by DFT calculations on CAIR and N{sup 5}-CAIR analogues in which the ribose 5'-phosphate is replaced with a methyl group. The calculations suggest that the nonaromatic tautomer of CAIR (isoCAIR) is only 3.1 kcal/mol higher in energy than its aromatic counterpart, implicating this species as a potential intermediate in the PurE-catalyzed reaction. A structure of wild-type PurE cocrystallized with 4-nitroaminoimidazole ribonucleotide (NO{sub 2}-AIR, a CAIR analogue) and structures of H45N and H45Q PurEs soaked with CAIR have been determined and provide the first insight into the binding of an intact PurE substrate. A comparison of 19 available structures of PurE and PurE mutants in apo and nucleotide-bound forms reveals a common, buried carboxylate or CO{sub 2} binding site for CAIR and N{sup 5}-CAIR in a hydrophobic pocket in whichmore » the carboxylate or CO{sub 2} interacts with backbone amides. This work has led to a mechanistic proposal in which the carboxylate orients the substrate for proton transfer from His45 to N{sup 5}-CAIR to form an enzyme-bound aminoimidazole ribonucleotide (AIR) and CO{sub 2} intermediate. Subsequent movement of the aminoimidazole moiety of AIR reorients it for addition of CO{sub 2} at C4 to generate isoCAIR. His45 is now in a position to remove a C4 proton to produce CAIR.« less

Authors:
; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930355
Report Number(s):
BNL-81074-2008-JA
Journal ID: ISSN 0006-2960; TRN: US0901373
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 46
Country of Publication:
United States
Language:
English
Subject:
72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS; AMIDES; AROMATICS; CATALYSIS; CRYSTALLOGRAPHY; ESCHERICHIA COLI; HISTIDINE; MUTAGENESIS; MUTANTS; NUCLEOTIDES; PROTONS; RIBOSE; SUBSTRATES; X-RAY DIFFRACTION; national synchrotron light source

Citation Formats

Hoskins,A., Morar, M., Kappack, T., Mathews, I., Zaugg, J., Barder, T., Peng, P., Okamoto, A., Ealick, S., and Stubbe, J. N5-CAIR Mutase: Role of a CO2 Binding Site and Substrate Movement in Catalysis. United States: N. p., 2007. Web. doi:10.1021/bi602436g.
Hoskins,A., Morar, M., Kappack, T., Mathews, I., Zaugg, J., Barder, T., Peng, P., Okamoto, A., Ealick, S., & Stubbe, J. N5-CAIR Mutase: Role of a CO2 Binding Site and Substrate Movement in Catalysis. United States. doi:10.1021/bi602436g.
Hoskins,A., Morar, M., Kappack, T., Mathews, I., Zaugg, J., Barder, T., Peng, P., Okamoto, A., Ealick, S., and Stubbe, J. Mon . "N5-CAIR Mutase: Role of a CO2 Binding Site and Substrate Movement in Catalysis". United States. doi:10.1021/bi602436g.
@article{osti_930355,
title = {N5-CAIR Mutase: Role of a CO2 Binding Site and Substrate Movement in Catalysis},
author = {Hoskins,A. and Morar, M. and Kappack, T. and Mathews, I. and Zaugg, J. and Barder, T. and Peng, P. and Okamoto, A. and Ealick, S. and Stubbe, J.},
abstractNote = {N{sup 5}-Carboxyaminoimidazole ribonucleotide mutase (N{sup 5}-CAIR mutase or PurE) from Escherichia coli catalyzes the reversible interconversion of N{sup 5}-CAIR to carboxyaminoimidazole ribonucleotide (CAIR) with direct CO{sub 2} transfer. Site-directed mutagenesis, a pH-rate profile, DFT calculations, and X-ray crystallography together provide new insight into the mechanism of this unusual transformation. These studies suggest that a conserved, protonated histidine (His45) plays an essential role in catalysis. The importance of proton transfers is supported by DFT calculations on CAIR and N{sup 5}-CAIR analogues in which the ribose 5'-phosphate is replaced with a methyl group. The calculations suggest that the nonaromatic tautomer of CAIR (isoCAIR) is only 3.1 kcal/mol higher in energy than its aromatic counterpart, implicating this species as a potential intermediate in the PurE-catalyzed reaction. A structure of wild-type PurE cocrystallized with 4-nitroaminoimidazole ribonucleotide (NO{sub 2}-AIR, a CAIR analogue) and structures of H45N and H45Q PurEs soaked with CAIR have been determined and provide the first insight into the binding of an intact PurE substrate. A comparison of 19 available structures of PurE and PurE mutants in apo and nucleotide-bound forms reveals a common, buried carboxylate or CO{sub 2} binding site for CAIR and N{sup 5}-CAIR in a hydrophobic pocket in which the carboxylate or CO{sub 2} interacts with backbone amides. This work has led to a mechanistic proposal in which the carboxylate orients the substrate for proton transfer from His45 to N{sup 5}-CAIR to form an enzyme-bound aminoimidazole ribonucleotide (AIR) and CO{sub 2} intermediate. Subsequent movement of the aminoimidazole moiety of AIR reorients it for addition of CO{sub 2} at C4 to generate isoCAIR. His45 is now in a position to remove a C4 proton to produce CAIR.},
doi = {10.1021/bi602436g},
journal = {Biochemistry},
number = ,
volume = 46,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • γ-Glutamyl transpeptidase 1 (GGT1) is a cell surface, N-terminal nucleophile hydrolase that cleaves glutathione and other γ-glutamyl compounds. GGT1 expression is essential in cysteine homeostasis, and its induction has been implicated in the pathology of asthma, reperfusion injury, and cancer. In this study, we report four new crystal structures of human GGT1 (hGGT1) that show conformational changes within the active site as the enzyme progresses from the free enzyme to inhibitor-bound tetrahedral transition states and finally to the glutamate-bound structure prior to the release of this final product of the reaction. The structure of the apoenzyme shows flexibility within themore » active site. The serine-borate-bound hGGT1 crystal structure demonstrates that serine-borate occupies the active site of the enzyme, resulting in an enzyme-inhibitor complex that replicates the enzyme's tetrahedral intermediate/transition state. The structure of GGsTop-bound hGGT1 reveals its interactions with the enzyme and why neutral phosphonate diesters are more potent inhibitors than monoanionic phosphonates. These structures are the first structures for any eukaryotic GGT that include a molecule in the active site covalently bound to the catalytic Thr-381. The glutamate-bound structure shows the conformation of the enzyme prior to release of the final product and reveals novel information regarding the displacement of the main chain atoms that form the oxyanion hole and movement of the lid loop region when the active site is occupied. Lastly,tThese data provide new insights into the mechanism of hGGT1-catalyzed reactions and will be invaluable in the development of new classes of hGGT1 inhibitors for therapeutic use.« less
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  • Galactofuranose (Galf) residues are present in cell wall glycoconjugates of numerous pathogenic microbes. Uridine 5{prime}-diphosphate (UDP) Galf, the biosynthetic precursor of Galf-containing glycoconjugates, is produced from UDP-galactopyranose (UDP-Galp) by the flavoenzyme UDP-galactopyranose mutase (UGM). The gene encoding UGM (glf) is essential for the viability of pathogens, including Mycobacterium tuberculosis, and this finding underscores the need to understand how UGM functions. Considerable effort has been devoted to elucidating the catalytic mechanism of UGM, but progress has been hindered by a lack of structural data for an enzyme-substrate complex. Such data could reveal not only substrate binding interactions but how UGM canmore » act preferentially on two very different substrates, UDP-Galp and UDP-Galf, yet avoid other structurally related UDP sugars present in the cell. Herein, we describe the first structure of a UGM-ligand complex, which provides insight into the catalytic mechanism and molecular basis for substrate selectivity. The structure of UGM from Klebsiella pneumoniae bound to the substrate analog UDP-glucose (UDP-Glc) was solved by X-ray crystallographic methods and refined to 2.5 {angstrom} resolution. The ligand is proximal to the cofactor, a finding that is consistent with a proposed mechanism in which the reduced flavin engages in covalent catalysis. Despite this proximity, the glucose ring of the substrate analog is positioned such that it disfavors covalent catalysis. This orientation is consistent with data indicating that UDP-Glc is not a substrate for UGM. The relative binding orientations of UDP-Galp and UDP-Glc were compared using saturation transfer difference NMR. The results indicate that the uridine moiety occupies a similar location in both ligand complexes, and this relevant binding mode is defined by our structural data. In contrast, the orientations of the glucose and galactose sugar moieties differ. To understand the consequences of these differences, we derived a model for the productive UGM-substrate complex that highlights interactions that can contribute to catalysis and substrate discrimination.« less