Setting the stage for electron transfer: Molecular basis of ABTS-binding to four laccases from Trametes versicolor at variable pH and protein oxidation state

Niels Johan Christensen, Kasper Planeta Kepp

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Laccases are multi-copper oxidases having exquisite oxidation power, high stability, and multiple industrial applications. Although Km varies ∼1000-fold across laccases, the molecular basis of substrate binding is poorly understood. Furthermore, laccase isoenzymes vary substantially in stability and activity for unknown reasons, and are thus useful probes of stability-function trade-offs relevant to protein engineering. We report here the first systematic comparison of ABTS-binding to different proteins, i.e. the four isoforms of Trametes versicolor, using a combination of sequence clustering, density functional theory calculations, homology modeling, and multiple induced-fit docking protocols at variable pH-dependent protonation states and T1-copper oxidation state. Clustering analysis provided a systematic overview of laccases across Trametes and revealed distinct isoenzyme classes (A–J) with the four T. versicolor isoforms belonging to separate classes. The T1 oxidation state had minor effect on ABTS binding, whereas the protonation state of Asp206 was important, consistent with site-directed mutagenesis studies. The absence of active poses for the δ-isoform agrees with its large Km, whereas the α-isoform with the smallest Km also had most active poses with protonated Asp206 corresponding to its pHopt∼2. Protonated Asp can bind to ABTS to form strong, active conformations partially explaining the low pHopt of fungal laccases toward ABTS. We identified several laccase properties optimal for ABTS binding. Notably, very high (R2∼0.99) correlation was observed between logKm (ABTS) and binding-pocket charge due to sites 157, 161, 269, 271, and 333, i.e. laccases optimal for ABTS turnover have positively charged anchor points in their pockets. Our work also demonstrates how activity-constraints can markedly improve docking to reduce inactive false positives.
Original languageEnglish
JournalJournal of Molecular Catalysis. B, Enzymatic
Volume100
Pages (from-to)68-77
ISSN1381-1177
DOIs
Publication statusPublished - 2014

Keywords

  • Laccase
  • Substrate
  • Sequence analysis
  • Docking
  • DFT

Cite this

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title = "Setting the stage for electron transfer: Molecular basis of ABTS-binding to four laccases from Trametes versicolor at variable pH and protein oxidation state",
abstract = "Laccases are multi-copper oxidases having exquisite oxidation power, high stability, and multiple industrial applications. Although Km varies ∼1000-fold across laccases, the molecular basis of substrate binding is poorly understood. Furthermore, laccase isoenzymes vary substantially in stability and activity for unknown reasons, and are thus useful probes of stability-function trade-offs relevant to protein engineering. We report here the first systematic comparison of ABTS-binding to different proteins, i.e. the four isoforms of Trametes versicolor, using a combination of sequence clustering, density functional theory calculations, homology modeling, and multiple induced-fit docking protocols at variable pH-dependent protonation states and T1-copper oxidation state. Clustering analysis provided a systematic overview of laccases across Trametes and revealed distinct isoenzyme classes (A–J) with the four T. versicolor isoforms belonging to separate classes. The T1 oxidation state had minor effect on ABTS binding, whereas the protonation state of Asp206 was important, consistent with site-directed mutagenesis studies. The absence of active poses for the δ-isoform agrees with its large Km, whereas the α-isoform with the smallest Km also had most active poses with protonated Asp206 corresponding to its pHopt∼2. Protonated Asp can bind to ABTS to form strong, active conformations partially explaining the low pHopt of fungal laccases toward ABTS. We identified several laccase properties optimal for ABTS binding. Notably, very high (R2∼0.99) correlation was observed between logKm (ABTS) and binding-pocket charge due to sites 157, 161, 269, 271, and 333, i.e. laccases optimal for ABTS turnover have positively charged anchor points in their pockets. Our work also demonstrates how activity-constraints can markedly improve docking to reduce inactive false positives.",
keywords = "Laccase, Substrate, Sequence analysis, Docking, DFT",
author = "Christensen, {Niels Johan} and Kepp, {Kasper Planeta}",
year = "2014",
doi = "10.1016/j.molcatb.2013.11.017",
language = "English",
volume = "100",
pages = "68--77",
journal = "Journal of Molecular Catalysis B: Enzymatic",
issn = "1381-1177",
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TY - JOUR

T1 - Setting the stage for electron transfer: Molecular basis of ABTS-binding to four laccases from Trametes versicolor at variable pH and protein oxidation state

AU - Christensen, Niels Johan

AU - Kepp, Kasper Planeta

PY - 2014

Y1 - 2014

N2 - Laccases are multi-copper oxidases having exquisite oxidation power, high stability, and multiple industrial applications. Although Km varies ∼1000-fold across laccases, the molecular basis of substrate binding is poorly understood. Furthermore, laccase isoenzymes vary substantially in stability and activity for unknown reasons, and are thus useful probes of stability-function trade-offs relevant to protein engineering. We report here the first systematic comparison of ABTS-binding to different proteins, i.e. the four isoforms of Trametes versicolor, using a combination of sequence clustering, density functional theory calculations, homology modeling, and multiple induced-fit docking protocols at variable pH-dependent protonation states and T1-copper oxidation state. Clustering analysis provided a systematic overview of laccases across Trametes and revealed distinct isoenzyme classes (A–J) with the four T. versicolor isoforms belonging to separate classes. The T1 oxidation state had minor effect on ABTS binding, whereas the protonation state of Asp206 was important, consistent with site-directed mutagenesis studies. The absence of active poses for the δ-isoform agrees with its large Km, whereas the α-isoform with the smallest Km also had most active poses with protonated Asp206 corresponding to its pHopt∼2. Protonated Asp can bind to ABTS to form strong, active conformations partially explaining the low pHopt of fungal laccases toward ABTS. We identified several laccase properties optimal for ABTS binding. Notably, very high (R2∼0.99) correlation was observed between logKm (ABTS) and binding-pocket charge due to sites 157, 161, 269, 271, and 333, i.e. laccases optimal for ABTS turnover have positively charged anchor points in their pockets. Our work also demonstrates how activity-constraints can markedly improve docking to reduce inactive false positives.

AB - Laccases are multi-copper oxidases having exquisite oxidation power, high stability, and multiple industrial applications. Although Km varies ∼1000-fold across laccases, the molecular basis of substrate binding is poorly understood. Furthermore, laccase isoenzymes vary substantially in stability and activity for unknown reasons, and are thus useful probes of stability-function trade-offs relevant to protein engineering. We report here the first systematic comparison of ABTS-binding to different proteins, i.e. the four isoforms of Trametes versicolor, using a combination of sequence clustering, density functional theory calculations, homology modeling, and multiple induced-fit docking protocols at variable pH-dependent protonation states and T1-copper oxidation state. Clustering analysis provided a systematic overview of laccases across Trametes and revealed distinct isoenzyme classes (A–J) with the four T. versicolor isoforms belonging to separate classes. The T1 oxidation state had minor effect on ABTS binding, whereas the protonation state of Asp206 was important, consistent with site-directed mutagenesis studies. The absence of active poses for the δ-isoform agrees with its large Km, whereas the α-isoform with the smallest Km also had most active poses with protonated Asp206 corresponding to its pHopt∼2. Protonated Asp can bind to ABTS to form strong, active conformations partially explaining the low pHopt of fungal laccases toward ABTS. We identified several laccase properties optimal for ABTS binding. Notably, very high (R2∼0.99) correlation was observed between logKm (ABTS) and binding-pocket charge due to sites 157, 161, 269, 271, and 333, i.e. laccases optimal for ABTS turnover have positively charged anchor points in their pockets. Our work also demonstrates how activity-constraints can markedly improve docking to reduce inactive false positives.

KW - Laccase

KW - Substrate

KW - Sequence analysis

KW - Docking

KW - DFT

U2 - 10.1016/j.molcatb.2013.11.017

DO - 10.1016/j.molcatb.2013.11.017

M3 - Journal article

VL - 100

SP - 68

EP - 77

JO - Journal of Molecular Catalysis B: Enzymatic

JF - Journal of Molecular Catalysis B: Enzymatic

SN - 1381-1177

ER -