NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions

Lytic polysaccharide monooxygenases currently classified as carbohydrate binding module family 33 (CBM33) and glycoside hydrolase family 61 (GH61) are likely to play important roles in future biorefining. However, the molecular basis of their unprecedented catalytic activity remains largely unknown....

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Veröffentlicht in:Proceedings of the National Academy of Sciences - PNAS 2012-11, Vol.109 (46), p.18779-18784
Hauptverfasser: Aachmann, Finn L, Sørlie, Morten, Skjåk-Bræk, Gudmund, Eijsink, Vincent G. H, Vaaje-Kolstad, Gustav
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container_issue 46
container_start_page 18779
container_title Proceedings of the National Academy of Sciences - PNAS
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creator Aachmann, Finn L
Sørlie, Morten
Skjåk-Bræk, Gudmund
Eijsink, Vincent G. H
Vaaje-Kolstad, Gustav
description Lytic polysaccharide monooxygenases currently classified as carbohydrate binding module family 33 (CBM33) and glycoside hydrolase family 61 (GH61) are likely to play important roles in future biorefining. However, the molecular basis of their unprecedented catalytic activity remains largely unknown. We have used NMR techniques and isothermal titration calorimetry to address structural and functional aspects of CBP21, a chitin-active CBM33. NMR structural and relaxation studies showed that CBP21 is a compact and rigid molecule, and the only exception is the catalytic metal binding site. NMR data further showed that His28 and His114 in the catalytic center bind a variety of divalent metal ions with a clear preference for Cu ²⁺ (K d = 55 nM; from isothermal titration calorimetry) and higher preference for Cu ¹⁺ (K d ∼ 1 nM; from the experimentally determined redox potential for CBP21-Cu ²⁺ of 275 mV using a thermodynamic cycle). Strong binding of Cu ¹⁺ was also reflected in a reduction in the p K ₐ values of the histidines by 3.6 and 2.2 pH units, respectively. Cyanide, a mimic of molecular oxygen, was found to bind to the metal ion only. These data support a model where copper is reduced on the enzyme by an externally provided electron and followed by oxygen binding and activation by internal electron transfer. Interactions of CBP21 with a crystalline substrate were mapped in a ²H/ ¹H exchange experiment, which showed that substrate binding involves an extended planar binding surface, including the metal binding site. Such a planar catalytic surface seems well-suited to interact with crystalline substrates.
doi_str_mv 10.1073/pnas.1208822109
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H</creatorcontrib><creatorcontrib>Vaaje-Kolstad, Gustav</creatorcontrib><title>NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>Lytic polysaccharide monooxygenases currently classified as carbohydrate binding module family 33 (CBM33) and glycoside hydrolase family 61 (GH61) are likely to play important roles in future biorefining. However, the molecular basis of their unprecedented catalytic activity remains largely unknown. We have used NMR techniques and isothermal titration calorimetry to address structural and functional aspects of CBP21, a chitin-active CBM33. NMR structural and relaxation studies showed that CBP21 is a compact and rigid molecule, and the only exception is the catalytic metal binding site. 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We have used NMR techniques and isothermal titration calorimetry to address structural and functional aspects of CBP21, a chitin-active CBM33. NMR structural and relaxation studies showed that CBP21 is a compact and rigid molecule, and the only exception is the catalytic metal binding site. NMR data further showed that His28 and His114 in the catalytic center bind a variety of divalent metal ions with a clear preference for Cu ²⁺ (K d = 55 nM; from isothermal titration calorimetry) and higher preference for Cu ¹⁺ (K d ∼ 1 nM; from the experimentally determined redox potential for CBP21-Cu ²⁺ of 275 mV using a thermodynamic cycle). Strong binding of Cu ¹⁺ was also reflected in a reduction in the p K ₐ values of the histidines by 3.6 and 2.2 pH units, respectively. Cyanide, a mimic of molecular oxygen, was found to bind to the metal ion only. 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subjects Bacterial Proteins - chemistry
Bacterial Proteins - metabolism
Binding sites
Biological Sciences
biorefining
calorimetry
carbohydrate binding
Carbohydrates
catalytic activity
Chitin
Copper
Copper - chemistry
Copper - metabolism
Crystal structure
Crystals
cyanides
Electron transfer
Enzymes
glycosides
histidine
Metal ions
Metalloproteins - chemistry
Metalloproteins - metabolism
Mixed Function Oxygenases - chemistry
Mixed Function Oxygenases - metabolism
NMR
Nuclear magnetic resonance
nuclear magnetic resonance spectroscopy
Nuclear Magnetic Resonance, Biomolecular
Oxygen
Polysaccharides
Protein Structure, Tertiary
Proteins
redox potential
Serratia marcescens - enzymology
Structure-Activity Relationship
Substrates
thermodynamics
Titration
title NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions
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