DNP NMR of carbohydrate converting enzymes

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Abstract

Dissolution dynamic nuclear polarization (DNP) NMR can be used to increase the sensitivity of 13CNMR signal by up to four orders of magnitude. This allows for real time monitoring of reactionsand observation of intermediates. The biggest drawback of the method is the loss of polarizationwith T1 relaxation, but even with this limitation, it is possible to obtain detailed reaction parametersin less than one minute. The enzyme investigated was β-galactosidase from E. coli (E.C. 3.2.1.23). It is well described and the mechanism is generally accepted to be a double displacement with a covalently bound intermediate, however, this evidence is based on mutant of X-ray crystallography and simulations. As the natural substrate lactose does not have any quaternary carbon with long T1, the unnatural substrate o-nitrophenyl β-D-galactopyranoside was used (figure 1) as the quaternarypositions have T1 relaxations of ca. 15 s instead of <2 s. The DNP NMR monitoring of the hydrolysis of this substrate can be seen in figure 2, and another use of this substrate is for optimizing the conditions for a labelled substrate (figure 1), which would further increase the signal and allow monitoring of the carbohydrate instead of the aglycon. This is, however, not commercially available and had to be synthesized from doubly labelled galactose.
Original languageEnglish
Publication date2017
Publication statusPublished - 2017
Event19th European Carbohydrate Symposium - CCIB, Barcelona, Spain
Duration: 2 Jul 20176 Jul 2017
Conference number: 19

Conference

Conference19th European Carbohydrate Symposium
Number19
LocationCCIB
CountrySpain
CityBarcelona
Period02/07/201706/07/2017

Cite this

Kjeldsen, C., Ardenkjær-Larsen, J. H., & Duus, J. Ø. (2017). DNP NMR of carbohydrate converting enzymes. Abstract from 19th European Carbohydrate Symposium, Barcelona, Spain.
@conference{1d81e201721d47539d43b2d25cc00dd7,
title = "DNP NMR of carbohydrate converting enzymes",
abstract = "Dissolution dynamic nuclear polarization (DNP) NMR can be used to increase the sensitivity of 13CNMR signal by up to four orders of magnitude. This allows for real time monitoring of reactionsand observation of intermediates. The biggest drawback of the method is the loss of polarizationwith T1 relaxation, but even with this limitation, it is possible to obtain detailed reaction parametersin less than one minute. The enzyme investigated was β-galactosidase from E. coli (E.C. 3.2.1.23). It is well described and the mechanism is generally accepted to be a double displacement with a covalently bound intermediate, however, this evidence is based on mutant of X-ray crystallography and simulations. As the natural substrate lactose does not have any quaternary carbon with long T1, the unnatural substrate o-nitrophenyl β-D-galactopyranoside was used (figure 1) as the quaternarypositions have T1 relaxations of ca. 15 s instead of <2 s. The DNP NMR monitoring of the hydrolysis of this substrate can be seen in figure 2, and another use of this substrate is for optimizing the conditions for a labelled substrate (figure 1), which would further increase the signal and allow monitoring of the carbohydrate instead of the aglycon. This is, however, not commercially available and had to be synthesized from doubly labelled galactose.",
author = "Christian Kjeldsen and Ardenkj{\ae}r-Larsen, {Jan Henrik} and Duus, {Jens {\O}llgaard}",
year = "2017",
language = "English",
note = "19th European Carbohydrate Symposium, EUROCARB ; Conference date: 02-07-2017 Through 06-07-2017",

}

Kjeldsen, C, Ardenkjær-Larsen, JH & Duus, JØ 2017, 'DNP NMR of carbohydrate converting enzymes' 19th European Carbohydrate Symposium, Barcelona, Spain, 02/07/2017 - 06/07/2017, .

DNP NMR of carbohydrate converting enzymes. / Kjeldsen, Christian; Ardenkjær-Larsen, Jan Henrik; Duus, Jens Øllgaard.

2017. Abstract from 19th European Carbohydrate Symposium, Barcelona, Spain.

Research output: Contribution to conferenceConference abstract for conferenceResearchpeer-review

TY - ABST

T1 - DNP NMR of carbohydrate converting enzymes

AU - Kjeldsen, Christian

AU - Ardenkjær-Larsen, Jan Henrik

AU - Duus, Jens Øllgaard

PY - 2017

Y1 - 2017

N2 - Dissolution dynamic nuclear polarization (DNP) NMR can be used to increase the sensitivity of 13CNMR signal by up to four orders of magnitude. This allows for real time monitoring of reactionsand observation of intermediates. The biggest drawback of the method is the loss of polarizationwith T1 relaxation, but even with this limitation, it is possible to obtain detailed reaction parametersin less than one minute. The enzyme investigated was β-galactosidase from E. coli (E.C. 3.2.1.23). It is well described and the mechanism is generally accepted to be a double displacement with a covalently bound intermediate, however, this evidence is based on mutant of X-ray crystallography and simulations. As the natural substrate lactose does not have any quaternary carbon with long T1, the unnatural substrate o-nitrophenyl β-D-galactopyranoside was used (figure 1) as the quaternarypositions have T1 relaxations of ca. 15 s instead of <2 s. The DNP NMR monitoring of the hydrolysis of this substrate can be seen in figure 2, and another use of this substrate is for optimizing the conditions for a labelled substrate (figure 1), which would further increase the signal and allow monitoring of the carbohydrate instead of the aglycon. This is, however, not commercially available and had to be synthesized from doubly labelled galactose.

AB - Dissolution dynamic nuclear polarization (DNP) NMR can be used to increase the sensitivity of 13CNMR signal by up to four orders of magnitude. This allows for real time monitoring of reactionsand observation of intermediates. The biggest drawback of the method is the loss of polarizationwith T1 relaxation, but even with this limitation, it is possible to obtain detailed reaction parametersin less than one minute. The enzyme investigated was β-galactosidase from E. coli (E.C. 3.2.1.23). It is well described and the mechanism is generally accepted to be a double displacement with a covalently bound intermediate, however, this evidence is based on mutant of X-ray crystallography and simulations. As the natural substrate lactose does not have any quaternary carbon with long T1, the unnatural substrate o-nitrophenyl β-D-galactopyranoside was used (figure 1) as the quaternarypositions have T1 relaxations of ca. 15 s instead of <2 s. The DNP NMR monitoring of the hydrolysis of this substrate can be seen in figure 2, and another use of this substrate is for optimizing the conditions for a labelled substrate (figure 1), which would further increase the signal and allow monitoring of the carbohydrate instead of the aglycon. This is, however, not commercially available and had to be synthesized from doubly labelled galactose.

M3 - Conference abstract for conference

ER -

Kjeldsen C, Ardenkjær-Larsen JH, Duus JØ. DNP NMR of carbohydrate converting enzymes. 2017. Abstract from 19th European Carbohydrate Symposium, Barcelona, Spain.