Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester substitution

Bijoy J. Desai, Yuki Goto, Alessandro Cembran, Alexander A. Fedorov, Steven C. Almo, Jiali Gao, Hiroaki Suga, John Alan Gerlt

Research output: Contribution to journalArticle

Abstract

Hydrogen bonds between backbone amide groups of enzymes and their substrates are often observed, but their importance in substrate binding and/or catalysis is not easy to investigate experimentally. We describe the generation and kinetic characterization of a backbone amide to ester substitution in the orotidine 5′-monophosphate (OMP) decarboxylase from Methanobacter thermoautotrophicum (MtOMPDC) to determine the importance of a backbone amide-substrate hydrogen bond. The MtOMPDC-catalyzed reaction is characterized by a rate enhancement (∼1017) that is among the largest for enzyme-catalyzed reactions. The reaction proceeds through a vinyl anion intermediate that may be stabilized by hydrogen bonding interaction between the backbone amide of a conserved active site serine residue (Ser-127) and oxygen (O4) of the pyrimidine moiety and/or electrostatic interactions with the conserved general acidic lysine (Lys-72). In vitro translation in conjunction with amber suppression using an orthogonal amber tRNA charged with L-glycerate (HOS) was used to generate the ester backbone substitution (S127HOS). With 5-fluoro OMP (FOMP) as substrate, the amide to ester substitution increased the value of Km by ∼1.5-fold and decreased the value of kcat by ∼50-fold. We conclude that (i) the hydrogen bond between the backbone amide of Ser-127 and O4 of the pyrimidine moiety contributes a modest factor (∼102) to the 1017 rate enhancement and (ii) the stabilization of the anionic intermediate is accomplished by electrostatic interactions, including its proximity of Lys-72. These conclusions are in good agreement with predictions obtained from hybrid quantum mechanical/molecular mechanical calculations.

Original languageEnglish (US)
Pages (from-to)15066-15071
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume111
Issue number42
DOIs
StatePublished - Oct 21 2014

Fingerprint

Carboxy-Lyases
Amides
Hydrogen
Esters
Amber
Static Electricity
Enzymes
Hydrogen Bonding
Transfer RNA
Catalysis
Serine
Lysine
Anions
orotidylic acid
Catalytic Domain
Oxygen

Keywords

  • Cell-free translation
  • Enzymology
  • Flexible tRNA acylation ribozyme
  • Unnatural protein residue

ASJC Scopus subject areas

  • General

Cite this

Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester substitution. / Desai, Bijoy J.; Goto, Yuki; Cembran, Alessandro; Fedorov, Alexander A.; Almo, Steven C.; Gao, Jiali; Suga, Hiroaki; Gerlt, John Alan.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 111, No. 42, 21.10.2014, p. 15066-15071.

Research output: Contribution to journalArticle

Desai, Bijoy J. ; Goto, Yuki ; Cembran, Alessandro ; Fedorov, Alexander A. ; Almo, Steven C. ; Gao, Jiali ; Suga, Hiroaki ; Gerlt, John Alan. / Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester substitution. In: Proceedings of the National Academy of Sciences of the United States of America. 2014 ; Vol. 111, No. 42. pp. 15066-15071.
@article{7ef042a6d84b4e1ca887fc28bf9bcd4b,
title = "Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester substitution",
abstract = "Hydrogen bonds between backbone amide groups of enzymes and their substrates are often observed, but their importance in substrate binding and/or catalysis is not easy to investigate experimentally. We describe the generation and kinetic characterization of a backbone amide to ester substitution in the orotidine 5′-monophosphate (OMP) decarboxylase from Methanobacter thermoautotrophicum (MtOMPDC) to determine the importance of a backbone amide-substrate hydrogen bond. The MtOMPDC-catalyzed reaction is characterized by a rate enhancement (∼1017) that is among the largest for enzyme-catalyzed reactions. The reaction proceeds through a vinyl anion intermediate that may be stabilized by hydrogen bonding interaction between the backbone amide of a conserved active site serine residue (Ser-127) and oxygen (O4) of the pyrimidine moiety and/or electrostatic interactions with the conserved general acidic lysine (Lys-72). In vitro translation in conjunction with amber suppression using an orthogonal amber tRNA charged with L-glycerate (HOS) was used to generate the ester backbone substitution (S127HOS). With 5-fluoro OMP (FOMP) as substrate, the amide to ester substitution increased the value of Km by ∼1.5-fold and decreased the value of kcat by ∼50-fold. We conclude that (i) the hydrogen bond between the backbone amide of Ser-127 and O4 of the pyrimidine moiety contributes a modest factor (∼102) to the 1017 rate enhancement and (ii) the stabilization of the anionic intermediate is accomplished by electrostatic interactions, including its proximity of Lys-72. These conclusions are in good agreement with predictions obtained from hybrid quantum mechanical/molecular mechanical calculations.",
keywords = "Cell-free translation, Enzymology, Flexible tRNA acylation ribozyme, Unnatural protein residue",
author = "Desai, {Bijoy J.} and Yuki Goto and Alessandro Cembran and Fedorov, {Alexander A.} and Almo, {Steven C.} and Jiali Gao and Hiroaki Suga and Gerlt, {John Alan}",
year = "2014",
month = "10",
day = "21",
doi = "10.1073/pnas.1411772111",
language = "English (US)",
volume = "111",
pages = "15066--15071",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "42",

}

TY - JOUR

T1 - Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester substitution

AU - Desai, Bijoy J.

AU - Goto, Yuki

AU - Cembran, Alessandro

AU - Fedorov, Alexander A.

AU - Almo, Steven C.

AU - Gao, Jiali

AU - Suga, Hiroaki

AU - Gerlt, John Alan

PY - 2014/10/21

Y1 - 2014/10/21

N2 - Hydrogen bonds between backbone amide groups of enzymes and their substrates are often observed, but their importance in substrate binding and/or catalysis is not easy to investigate experimentally. We describe the generation and kinetic characterization of a backbone amide to ester substitution in the orotidine 5′-monophosphate (OMP) decarboxylase from Methanobacter thermoautotrophicum (MtOMPDC) to determine the importance of a backbone amide-substrate hydrogen bond. The MtOMPDC-catalyzed reaction is characterized by a rate enhancement (∼1017) that is among the largest for enzyme-catalyzed reactions. The reaction proceeds through a vinyl anion intermediate that may be stabilized by hydrogen bonding interaction between the backbone amide of a conserved active site serine residue (Ser-127) and oxygen (O4) of the pyrimidine moiety and/or electrostatic interactions with the conserved general acidic lysine (Lys-72). In vitro translation in conjunction with amber suppression using an orthogonal amber tRNA charged with L-glycerate (HOS) was used to generate the ester backbone substitution (S127HOS). With 5-fluoro OMP (FOMP) as substrate, the amide to ester substitution increased the value of Km by ∼1.5-fold and decreased the value of kcat by ∼50-fold. We conclude that (i) the hydrogen bond between the backbone amide of Ser-127 and O4 of the pyrimidine moiety contributes a modest factor (∼102) to the 1017 rate enhancement and (ii) the stabilization of the anionic intermediate is accomplished by electrostatic interactions, including its proximity of Lys-72. These conclusions are in good agreement with predictions obtained from hybrid quantum mechanical/molecular mechanical calculations.

AB - Hydrogen bonds between backbone amide groups of enzymes and their substrates are often observed, but their importance in substrate binding and/or catalysis is not easy to investigate experimentally. We describe the generation and kinetic characterization of a backbone amide to ester substitution in the orotidine 5′-monophosphate (OMP) decarboxylase from Methanobacter thermoautotrophicum (MtOMPDC) to determine the importance of a backbone amide-substrate hydrogen bond. The MtOMPDC-catalyzed reaction is characterized by a rate enhancement (∼1017) that is among the largest for enzyme-catalyzed reactions. The reaction proceeds through a vinyl anion intermediate that may be stabilized by hydrogen bonding interaction between the backbone amide of a conserved active site serine residue (Ser-127) and oxygen (O4) of the pyrimidine moiety and/or electrostatic interactions with the conserved general acidic lysine (Lys-72). In vitro translation in conjunction with amber suppression using an orthogonal amber tRNA charged with L-glycerate (HOS) was used to generate the ester backbone substitution (S127HOS). With 5-fluoro OMP (FOMP) as substrate, the amide to ester substitution increased the value of Km by ∼1.5-fold and decreased the value of kcat by ∼50-fold. We conclude that (i) the hydrogen bond between the backbone amide of Ser-127 and O4 of the pyrimidine moiety contributes a modest factor (∼102) to the 1017 rate enhancement and (ii) the stabilization of the anionic intermediate is accomplished by electrostatic interactions, including its proximity of Lys-72. These conclusions are in good agreement with predictions obtained from hybrid quantum mechanical/molecular mechanical calculations.

KW - Cell-free translation

KW - Enzymology

KW - Flexible tRNA acylation ribozyme

KW - Unnatural protein residue

UR - http://www.scopus.com/inward/record.url?scp=84908065770&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84908065770&partnerID=8YFLogxK

U2 - 10.1073/pnas.1411772111

DO - 10.1073/pnas.1411772111

M3 - Article

VL - 111

SP - 15066

EP - 15071

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 42

ER -