The mechanism of the reaction catalyzed by uronate isomerase illustrates how an isomerase may have evolved from a hydrolase within the amidohydrolase superfamily

Tinh T. Nguyen, Alexander A. Fedorov, LaKenya Williams, Elena V. Fedorov, Yingchun Li, Chengfu Xu, Steven C. Almo, Frank M. Raushel

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Uronate isomerase (URI) catalyzes the reversible isomerization of D-glucuronate to D-fructuronate and of D-galacturonate to D-tagaturonate. URI is a member of the amidohydrolase superfamily (AHS), a highly divergent group of enzymes that catalyze primarily hydrolytic reactions. The chemical mechanism and active site structure of URI were investigated in an attempt to improve our understanding of how an active site template that apparently evolved to catalyze hydrolytic reactions has been reforged to catalyze an isomerization reaction. The pH-rate profiles for kcat and kcat/Km for URI from Escherichia coli are bell-shaped and indicate that one group must be unprotonated and another residue must be protonated for catalytic activity. Primary isotope effects on the kinetic constants with [2-2H]-D- glucuronate and the effects of changes in solvent viscosity are consistent with product release being the rate-limiting step. The X-ray structure of Bh0493, a URI from Bacillus halodurans, was determined in the presence of the substrate D-glucuronate. The bound complex showed that the mononuclear metal center in the active site is ligated to the C-6 carboxylate and the C-5 hydroxyl group of the substrate. This hydroxyl group is also hydrogen bonded to Asp-355 in the same orientation as the hydroxide or water is bound in those members of the AHS that catalyze hydrolytic reactions. In addition, the C-2 and C-3 hydroxyl groups of the substrate are hydrogen bonded to Arg-357 and the carbonyl group at C-1 is hydrogen bonded to Tyr-50. A chemical mechanism is proposed that utilizes a proton transfer from C-2 of D-glucuronate to C-1 that is initiated by the combined actions of Asp-355 from the end of β-strand 8 and the C-5 hydroxyl of the substrate that is bound to the metal ion. The formation of the proposed cis-enediol intermediate is further facilitated by the shuttling of the proton between the C-2 and C-1 oxygens by the conserved Tyr-50 and/or Arg-355.

Original languageEnglish (US)
Pages (from-to)8879-8890
Number of pages12
JournalBiochemistry
Volume48
Issue number37
DOIs
StatePublished - Sep 22 2009

Fingerprint

Amidohydrolases
Isomerases
Hydrolases
Glucuronic Acid
Hydroxyl Radical
Hydrogen
Catalytic Domain
Substrates
Isomerization
Protons
Metals
Proton transfer
Bacilli
Viscosity
Isotopes
Escherichia coli
Bacillus
Metal ions
Catalyst activity
X-Rays

ASJC Scopus subject areas

  • Biochemistry

Cite this

The mechanism of the reaction catalyzed by uronate isomerase illustrates how an isomerase may have evolved from a hydrolase within the amidohydrolase superfamily. / Nguyen, Tinh T.; Fedorov, Alexander A.; Williams, LaKenya; Fedorov, Elena V.; Li, Yingchun; Xu, Chengfu; Almo, Steven C.; Raushel, Frank M.

In: Biochemistry, Vol. 48, No. 37, 22.09.2009, p. 8879-8890.

Research output: Contribution to journalArticle

Nguyen, Tinh T. ; Fedorov, Alexander A. ; Williams, LaKenya ; Fedorov, Elena V. ; Li, Yingchun ; Xu, Chengfu ; Almo, Steven C. ; Raushel, Frank M. / The mechanism of the reaction catalyzed by uronate isomerase illustrates how an isomerase may have evolved from a hydrolase within the amidohydrolase superfamily. In: Biochemistry. 2009 ; Vol. 48, No. 37. pp. 8879-8890.
@article{a354fb021f3b4099923637f25e4b7d60,
title = "The mechanism of the reaction catalyzed by uronate isomerase illustrates how an isomerase may have evolved from a hydrolase within the amidohydrolase superfamily",
abstract = "Uronate isomerase (URI) catalyzes the reversible isomerization of D-glucuronate to D-fructuronate and of D-galacturonate to D-tagaturonate. URI is a member of the amidohydrolase superfamily (AHS), a highly divergent group of enzymes that catalyze primarily hydrolytic reactions. The chemical mechanism and active site structure of URI were investigated in an attempt to improve our understanding of how an active site template that apparently evolved to catalyze hydrolytic reactions has been reforged to catalyze an isomerization reaction. The pH-rate profiles for kcat and kcat/Km for URI from Escherichia coli are bell-shaped and indicate that one group must be unprotonated and another residue must be protonated for catalytic activity. Primary isotope effects on the kinetic constants with [2-2H]-D- glucuronate and the effects of changes in solvent viscosity are consistent with product release being the rate-limiting step. The X-ray structure of Bh0493, a URI from Bacillus halodurans, was determined in the presence of the substrate D-glucuronate. The bound complex showed that the mononuclear metal center in the active site is ligated to the C-6 carboxylate and the C-5 hydroxyl group of the substrate. This hydroxyl group is also hydrogen bonded to Asp-355 in the same orientation as the hydroxide or water is bound in those members of the AHS that catalyze hydrolytic reactions. In addition, the C-2 and C-3 hydroxyl groups of the substrate are hydrogen bonded to Arg-357 and the carbonyl group at C-1 is hydrogen bonded to Tyr-50. A chemical mechanism is proposed that utilizes a proton transfer from C-2 of D-glucuronate to C-1 that is initiated by the combined actions of Asp-355 from the end of β-strand 8 and the C-5 hydroxyl of the substrate that is bound to the metal ion. The formation of the proposed cis-enediol intermediate is further facilitated by the shuttling of the proton between the C-2 and C-1 oxygens by the conserved Tyr-50 and/or Arg-355.",
author = "Nguyen, {Tinh T.} and Fedorov, {Alexander A.} and LaKenya Williams and Fedorov, {Elena V.} and Yingchun Li and Chengfu Xu and Almo, {Steven C.} and Raushel, {Frank M.}",
year = "2009",
month = "9",
day = "22",
doi = "10.1021/bi901046x",
language = "English (US)",
volume = "48",
pages = "8879--8890",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "37",

}

TY - JOUR

T1 - The mechanism of the reaction catalyzed by uronate isomerase illustrates how an isomerase may have evolved from a hydrolase within the amidohydrolase superfamily

AU - Nguyen, Tinh T.

AU - Fedorov, Alexander A.

AU - Williams, LaKenya

AU - Fedorov, Elena V.

AU - Li, Yingchun

AU - Xu, Chengfu

AU - Almo, Steven C.

AU - Raushel, Frank M.

PY - 2009/9/22

Y1 - 2009/9/22

N2 - Uronate isomerase (URI) catalyzes the reversible isomerization of D-glucuronate to D-fructuronate and of D-galacturonate to D-tagaturonate. URI is a member of the amidohydrolase superfamily (AHS), a highly divergent group of enzymes that catalyze primarily hydrolytic reactions. The chemical mechanism and active site structure of URI were investigated in an attempt to improve our understanding of how an active site template that apparently evolved to catalyze hydrolytic reactions has been reforged to catalyze an isomerization reaction. The pH-rate profiles for kcat and kcat/Km for URI from Escherichia coli are bell-shaped and indicate that one group must be unprotonated and another residue must be protonated for catalytic activity. Primary isotope effects on the kinetic constants with [2-2H]-D- glucuronate and the effects of changes in solvent viscosity are consistent with product release being the rate-limiting step. The X-ray structure of Bh0493, a URI from Bacillus halodurans, was determined in the presence of the substrate D-glucuronate. The bound complex showed that the mononuclear metal center in the active site is ligated to the C-6 carboxylate and the C-5 hydroxyl group of the substrate. This hydroxyl group is also hydrogen bonded to Asp-355 in the same orientation as the hydroxide or water is bound in those members of the AHS that catalyze hydrolytic reactions. In addition, the C-2 and C-3 hydroxyl groups of the substrate are hydrogen bonded to Arg-357 and the carbonyl group at C-1 is hydrogen bonded to Tyr-50. A chemical mechanism is proposed that utilizes a proton transfer from C-2 of D-glucuronate to C-1 that is initiated by the combined actions of Asp-355 from the end of β-strand 8 and the C-5 hydroxyl of the substrate that is bound to the metal ion. The formation of the proposed cis-enediol intermediate is further facilitated by the shuttling of the proton between the C-2 and C-1 oxygens by the conserved Tyr-50 and/or Arg-355.

AB - Uronate isomerase (URI) catalyzes the reversible isomerization of D-glucuronate to D-fructuronate and of D-galacturonate to D-tagaturonate. URI is a member of the amidohydrolase superfamily (AHS), a highly divergent group of enzymes that catalyze primarily hydrolytic reactions. The chemical mechanism and active site structure of URI were investigated in an attempt to improve our understanding of how an active site template that apparently evolved to catalyze hydrolytic reactions has been reforged to catalyze an isomerization reaction. The pH-rate profiles for kcat and kcat/Km for URI from Escherichia coli are bell-shaped and indicate that one group must be unprotonated and another residue must be protonated for catalytic activity. Primary isotope effects on the kinetic constants with [2-2H]-D- glucuronate and the effects of changes in solvent viscosity are consistent with product release being the rate-limiting step. The X-ray structure of Bh0493, a URI from Bacillus halodurans, was determined in the presence of the substrate D-glucuronate. The bound complex showed that the mononuclear metal center in the active site is ligated to the C-6 carboxylate and the C-5 hydroxyl group of the substrate. This hydroxyl group is also hydrogen bonded to Asp-355 in the same orientation as the hydroxide or water is bound in those members of the AHS that catalyze hydrolytic reactions. In addition, the C-2 and C-3 hydroxyl groups of the substrate are hydrogen bonded to Arg-357 and the carbonyl group at C-1 is hydrogen bonded to Tyr-50. A chemical mechanism is proposed that utilizes a proton transfer from C-2 of D-glucuronate to C-1 that is initiated by the combined actions of Asp-355 from the end of β-strand 8 and the C-5 hydroxyl of the substrate that is bound to the metal ion. The formation of the proposed cis-enediol intermediate is further facilitated by the shuttling of the proton between the C-2 and C-1 oxygens by the conserved Tyr-50 and/or Arg-355.

UR - http://www.scopus.com/inward/record.url?scp=70349110534&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=70349110534&partnerID=8YFLogxK

U2 - 10.1021/bi901046x

DO - 10.1021/bi901046x

M3 - Article

VL - 48

SP - 8879

EP - 8890

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 37

ER -