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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}
}