Mutation of Tyr-218 to Phe in Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase: Effects on bioelectronic interface performance

Brian L. Hassler, Megan Dennis, Maris Laivenieks, J. Gregory Zeikus, Robert M. Worden

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Bioelectronic interfaces that facilitate electron transfer between the electrode and a dehydrogenase enzyme have potential applications in biosensors, biocatalytic reactors, and biological fuel cells. The secondary alcohol dehydrogenase (2° ADH) from Thermoanaerobacter ethanolicus is especially well suited for the development of such bioelectronic interfaces because of its thermostability and facile production and purification. However, the natural cofactor for the enzyme, β-nicotinamide adenine dinucleotide phosphate (NADP+), is more expensive and less stable than β-nicotinamide adenine dinucleotide (NAD+). PCR-based, site-directed mutagenesis was performed on 2° ADH in an attempt to adjust the cofactor specificity toward NAD+ by mutating Tyr218 to Phe (Y218F 2° ADH). This mutation increased the K m(app) for NADP+ 200-fold while decreasing the K m(app) for NAD+ 2.5-fold. The mutant enzyme was incorporated into a bioelectronic interface that established electrical communication between the enzyme, the NAD+, the electron mediator toluidine blue O (TBO), and a gold electrode. Cyclic voltammetry, impedance spectroscopy, gas chromatography, mass spectrometry, constant potential amperometry, and chronoamperometry were used to characterize the mutant and wild-type enzyme incorporated in the bioelectronic interface. The Y218F 2° ADH exhibited a fourfold increase in the turnover ratio compared to the wild type in the presence of NAD+. The electrochemical and kinetic measurements support the prediction that the Rossmann fold of the enzyme binds to the phosphate moiety of the cofactor. During the 45 min of continuous operation, NAD+ was electrically recycled 6.7×104 times, suggesting that the Y218F 2° ADH-modified bioelectronic interface is stable.

Original languageEnglish (US)
Pages (from-to)1-15
Number of pages15
JournalApplied Biochemistry and Biotechnology
Volume143
Issue number1
DOIs
StatePublished - Oct 1 2007
Externally publishedYes

Fingerprint

Thermoanaerobacter
NAD
Alcohols
Enzymes
Mutation
NADP
Application programs
Phosphates
Biological fuel cells
Electrodes
Mutagenesis
Chronoamperometry
Electrons
Dielectric Spectroscopy
Tolonium Chloride
Biosensors
Coenzymes
Gas chromatography
Biosensing Techniques
Cyclic voltammetry

Keywords

  • Biocatalysis
  • Bioelectronic
  • Biosensor
  • Cofactor regeneration
  • Electron mediator
  • NAD
  • NADP
  • Secondary alcohol dehydrogenase
  • Site-directed mutagenesis
  • Toluidine blue

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology

Cite this

Mutation of Tyr-218 to Phe in Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase : Effects on bioelectronic interface performance. / Hassler, Brian L.; Dennis, Megan; Laivenieks, Maris; Zeikus, J. Gregory; Worden, Robert M.

In: Applied Biochemistry and Biotechnology, Vol. 143, No. 1, 01.10.2007, p. 1-15.

Research output: Contribution to journalArticle

@article{897dd995c4fe4a15a1a60c74d136c55c,
title = "Mutation of Tyr-218 to Phe in Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase: Effects on bioelectronic interface performance",
abstract = "Bioelectronic interfaces that facilitate electron transfer between the electrode and a dehydrogenase enzyme have potential applications in biosensors, biocatalytic reactors, and biological fuel cells. The secondary alcohol dehydrogenase (2° ADH) from Thermoanaerobacter ethanolicus is especially well suited for the development of such bioelectronic interfaces because of its thermostability and facile production and purification. However, the natural cofactor for the enzyme, β-nicotinamide adenine dinucleotide phosphate (NADP+), is more expensive and less stable than β-nicotinamide adenine dinucleotide (NAD+). PCR-based, site-directed mutagenesis was performed on 2° ADH in an attempt to adjust the cofactor specificity toward NAD+ by mutating Tyr218 to Phe (Y218F 2° ADH). This mutation increased the K m(app) for NADP+ 200-fold while decreasing the K m(app) for NAD+ 2.5-fold. The mutant enzyme was incorporated into a bioelectronic interface that established electrical communication between the enzyme, the NAD+, the electron mediator toluidine blue O (TBO), and a gold electrode. Cyclic voltammetry, impedance spectroscopy, gas chromatography, mass spectrometry, constant potential amperometry, and chronoamperometry were used to characterize the mutant and wild-type enzyme incorporated in the bioelectronic interface. The Y218F 2° ADH exhibited a fourfold increase in the turnover ratio compared to the wild type in the presence of NAD+. The electrochemical and kinetic measurements support the prediction that the Rossmann fold of the enzyme binds to the phosphate moiety of the cofactor. During the 45 min of continuous operation, NAD+ was electrically recycled 6.7×104 times, suggesting that the Y218F 2° ADH-modified bioelectronic interface is stable.",
keywords = "Biocatalysis, Bioelectronic, Biosensor, Cofactor regeneration, Electron mediator, NAD, NADP, Secondary alcohol dehydrogenase, Site-directed mutagenesis, Toluidine blue",
author = "Hassler, {Brian L.} and Megan Dennis and Maris Laivenieks and Zeikus, {J. Gregory} and Worden, {Robert M.}",
year = "2007",
month = "10",
day = "1",
doi = "10.1007/s12010-007-0027-2",
language = "English (US)",
volume = "143",
pages = "1--15",
journal = "Applied Biochemistry and Biotechnology - Part A Enzyme Engineering and Biotechnology",
issn = "0273-2289",
publisher = "Humana Press",
number = "1",

}

TY - JOUR

T1 - Mutation of Tyr-218 to Phe in Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase

T2 - Effects on bioelectronic interface performance

AU - Hassler, Brian L.

AU - Dennis, Megan

AU - Laivenieks, Maris

AU - Zeikus, J. Gregory

AU - Worden, Robert M.

PY - 2007/10/1

Y1 - 2007/10/1

N2 - Bioelectronic interfaces that facilitate electron transfer between the electrode and a dehydrogenase enzyme have potential applications in biosensors, biocatalytic reactors, and biological fuel cells. The secondary alcohol dehydrogenase (2° ADH) from Thermoanaerobacter ethanolicus is especially well suited for the development of such bioelectronic interfaces because of its thermostability and facile production and purification. However, the natural cofactor for the enzyme, β-nicotinamide adenine dinucleotide phosphate (NADP+), is more expensive and less stable than β-nicotinamide adenine dinucleotide (NAD+). PCR-based, site-directed mutagenesis was performed on 2° ADH in an attempt to adjust the cofactor specificity toward NAD+ by mutating Tyr218 to Phe (Y218F 2° ADH). This mutation increased the K m(app) for NADP+ 200-fold while decreasing the K m(app) for NAD+ 2.5-fold. The mutant enzyme was incorporated into a bioelectronic interface that established electrical communication between the enzyme, the NAD+, the electron mediator toluidine blue O (TBO), and a gold electrode. Cyclic voltammetry, impedance spectroscopy, gas chromatography, mass spectrometry, constant potential amperometry, and chronoamperometry were used to characterize the mutant and wild-type enzyme incorporated in the bioelectronic interface. The Y218F 2° ADH exhibited a fourfold increase in the turnover ratio compared to the wild type in the presence of NAD+. The electrochemical and kinetic measurements support the prediction that the Rossmann fold of the enzyme binds to the phosphate moiety of the cofactor. During the 45 min of continuous operation, NAD+ was electrically recycled 6.7×104 times, suggesting that the Y218F 2° ADH-modified bioelectronic interface is stable.

AB - Bioelectronic interfaces that facilitate electron transfer between the electrode and a dehydrogenase enzyme have potential applications in biosensors, biocatalytic reactors, and biological fuel cells. The secondary alcohol dehydrogenase (2° ADH) from Thermoanaerobacter ethanolicus is especially well suited for the development of such bioelectronic interfaces because of its thermostability and facile production and purification. However, the natural cofactor for the enzyme, β-nicotinamide adenine dinucleotide phosphate (NADP+), is more expensive and less stable than β-nicotinamide adenine dinucleotide (NAD+). PCR-based, site-directed mutagenesis was performed on 2° ADH in an attempt to adjust the cofactor specificity toward NAD+ by mutating Tyr218 to Phe (Y218F 2° ADH). This mutation increased the K m(app) for NADP+ 200-fold while decreasing the K m(app) for NAD+ 2.5-fold. The mutant enzyme was incorporated into a bioelectronic interface that established electrical communication between the enzyme, the NAD+, the electron mediator toluidine blue O (TBO), and a gold electrode. Cyclic voltammetry, impedance spectroscopy, gas chromatography, mass spectrometry, constant potential amperometry, and chronoamperometry were used to characterize the mutant and wild-type enzyme incorporated in the bioelectronic interface. The Y218F 2° ADH exhibited a fourfold increase in the turnover ratio compared to the wild type in the presence of NAD+. The electrochemical and kinetic measurements support the prediction that the Rossmann fold of the enzyme binds to the phosphate moiety of the cofactor. During the 45 min of continuous operation, NAD+ was electrically recycled 6.7×104 times, suggesting that the Y218F 2° ADH-modified bioelectronic interface is stable.

KW - Biocatalysis

KW - Bioelectronic

KW - Biosensor

KW - Cofactor regeneration

KW - Electron mediator

KW - NAD

KW - NADP

KW - Secondary alcohol dehydrogenase

KW - Site-directed mutagenesis

KW - Toluidine blue

UR - http://www.scopus.com/inward/record.url?scp=36949022904&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=36949022904&partnerID=8YFLogxK

U2 - 10.1007/s12010-007-0027-2

DO - 10.1007/s12010-007-0027-2

M3 - Article

C2 - 18025592

AN - SCOPUS:36949022904

VL - 143

SP - 1

EP - 15

JO - Applied Biochemistry and Biotechnology - Part A Enzyme Engineering and Biotechnology

JF - Applied Biochemistry and Biotechnology - Part A Enzyme Engineering and Biotechnology

SN - 0273-2289

IS - 1

ER -