Molecular Characterization of an NADPH-Dependent Acetoin Reductase/2,3-Butanediol Dehydrogenase from Clostridium beijerinckii NCIMB 8052

J.G.J. Raedts, M.A.J. Siemerink, M. Levisson, J. van der Oost, S.W.M. Kengen

Research output: Contribution to journalArticleAcademicpeer-review

16 Citations (Scopus)

Abstract

Acetoin reductase is an important enzyme for the fermentative production of 2,3-butanediol, a chemical compound with a very broad industrial use. Here, we report on the discovery and characterization of an acetoin reductase from Clostridium beijerinckii NCIMB 8052. An in silico screen of the C. beijerinckii genome revealed eight potential acetoin reductases. One of them (CBEI_1464) showed substantial acetoin reductase activity after expression in Escherichia coli. The purified enzyme (C. beijerinckii acetoin reductase [Cb-ACR]) was found to exist predominantly as a homodimer. In addition to acetoin (or 2,3-butanediol), other secondary alcohols and corresponding ketones were converted as well, provided that another electronegative group was attached to the adjacent C-3 carbon. Optimal activity was at pH 6.5 (reduction) and 9.5 (oxidation) and around 68°C. Cb-ACR accepts both NADH and NADPH as electron donors; however, unlike closely related enzymes, NADPH is preferred (Km, 32 µM). Cb-ACR was compared to characterized close homologs, all belonging to the "threonine dehydrogenase and related Zn-dependent dehydrogenases" (COG1063). Metal analysis confirmed the presence of 2 Zn(2+) atoms. To gain insight into the substrate and cofactor specificity, a structural model was constructed. The catalytic zinc atom is likely coordinated by Cys37, His70, and Glu71, while the structural zinc site is probably composed of Cys100, Cys103, Cys106, and Cys114. Residues determining NADP specificity were predicted as well. The physiological role of Cb-ACR in C. beijerinckii is discussed
Original languageEnglish
Pages (from-to)2011-2020
JournalApplied and Environmental Microbiology
Volume80
Issue number6
DOIs
Publication statusPublished - 2014

Fingerprint

butanediol dehydrogenase
Clostridium beijerinckii
butanediol
acetoin
NADP
NADP (coenzyme)
enzyme
zinc
chemical compound
ketone
alcohol
L-threonine 3-dehydrogenase
genome
Zinc
oxidation
substrate
electron
Enzymes
metal
carbon

Keywords

  • l-threonine dehydrogenase
  • protein-structure prediction
  • crystal-structure
  • alcohol dehydrogenases
  • (2r,3r)-2,3-butanediol dehydrogenase
  • thermoanaerobacter-brockii
  • escherichia-coli
  • meso-2,3-butanediol dehydrogenase
  • sulfolobus-solfataricus
  • butanol fer

Cite this

@article{c3c1aadb338f46f5bfc13bd59b07f64f,
title = "Molecular Characterization of an NADPH-Dependent Acetoin Reductase/2,3-Butanediol Dehydrogenase from Clostridium beijerinckii NCIMB 8052",
abstract = "Acetoin reductase is an important enzyme for the fermentative production of 2,3-butanediol, a chemical compound with a very broad industrial use. Here, we report on the discovery and characterization of an acetoin reductase from Clostridium beijerinckii NCIMB 8052. An in silico screen of the C. beijerinckii genome revealed eight potential acetoin reductases. One of them (CBEI_1464) showed substantial acetoin reductase activity after expression in Escherichia coli. The purified enzyme (C. beijerinckii acetoin reductase [Cb-ACR]) was found to exist predominantly as a homodimer. In addition to acetoin (or 2,3-butanediol), other secondary alcohols and corresponding ketones were converted as well, provided that another electronegative group was attached to the adjacent C-3 carbon. Optimal activity was at pH 6.5 (reduction) and 9.5 (oxidation) and around 68°C. Cb-ACR accepts both NADH and NADPH as electron donors; however, unlike closely related enzymes, NADPH is preferred (Km, 32 µM). Cb-ACR was compared to characterized close homologs, all belonging to the {"}threonine dehydrogenase and related Zn-dependent dehydrogenases{"} (COG1063). Metal analysis confirmed the presence of 2 Zn(2+) atoms. To gain insight into the substrate and cofactor specificity, a structural model was constructed. The catalytic zinc atom is likely coordinated by Cys37, His70, and Glu71, while the structural zinc site is probably composed of Cys100, Cys103, Cys106, and Cys114. Residues determining NADP specificity were predicted as well. The physiological role of Cb-ACR in C. beijerinckii is discussed",
keywords = "l-threonine dehydrogenase, protein-structure prediction, crystal-structure, alcohol dehydrogenases, (2r,3r)-2,3-butanediol dehydrogenase, thermoanaerobacter-brockii, escherichia-coli, meso-2,3-butanediol dehydrogenase, sulfolobus-solfataricus, butanol fer",
author = "J.G.J. Raedts and M.A.J. Siemerink and M. Levisson and {van der Oost}, J. and S.W.M. Kengen",
year = "2014",
doi = "10.1128/AEM.04007-13",
language = "English",
volume = "80",
pages = "2011--2020",
journal = "Applied and Environmental Microbiology",
issn = "0099-2240",
publisher = "American Society for Microbiology",
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}

Molecular Characterization of an NADPH-Dependent Acetoin Reductase/2,3-Butanediol Dehydrogenase from Clostridium beijerinckii NCIMB 8052. / Raedts, J.G.J.; Siemerink, M.A.J.; Levisson, M.; van der Oost, J.; Kengen, S.W.M.

In: Applied and Environmental Microbiology, Vol. 80, No. 6, 2014, p. 2011-2020.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Molecular Characterization of an NADPH-Dependent Acetoin Reductase/2,3-Butanediol Dehydrogenase from Clostridium beijerinckii NCIMB 8052

AU - Raedts, J.G.J.

AU - Siemerink, M.A.J.

AU - Levisson, M.

AU - van der Oost, J.

AU - Kengen, S.W.M.

PY - 2014

Y1 - 2014

N2 - Acetoin reductase is an important enzyme for the fermentative production of 2,3-butanediol, a chemical compound with a very broad industrial use. Here, we report on the discovery and characterization of an acetoin reductase from Clostridium beijerinckii NCIMB 8052. An in silico screen of the C. beijerinckii genome revealed eight potential acetoin reductases. One of them (CBEI_1464) showed substantial acetoin reductase activity after expression in Escherichia coli. The purified enzyme (C. beijerinckii acetoin reductase [Cb-ACR]) was found to exist predominantly as a homodimer. In addition to acetoin (or 2,3-butanediol), other secondary alcohols and corresponding ketones were converted as well, provided that another electronegative group was attached to the adjacent C-3 carbon. Optimal activity was at pH 6.5 (reduction) and 9.5 (oxidation) and around 68°C. Cb-ACR accepts both NADH and NADPH as electron donors; however, unlike closely related enzymes, NADPH is preferred (Km, 32 µM). Cb-ACR was compared to characterized close homologs, all belonging to the "threonine dehydrogenase and related Zn-dependent dehydrogenases" (COG1063). Metal analysis confirmed the presence of 2 Zn(2+) atoms. To gain insight into the substrate and cofactor specificity, a structural model was constructed. The catalytic zinc atom is likely coordinated by Cys37, His70, and Glu71, while the structural zinc site is probably composed of Cys100, Cys103, Cys106, and Cys114. Residues determining NADP specificity were predicted as well. The physiological role of Cb-ACR in C. beijerinckii is discussed

AB - Acetoin reductase is an important enzyme for the fermentative production of 2,3-butanediol, a chemical compound with a very broad industrial use. Here, we report on the discovery and characterization of an acetoin reductase from Clostridium beijerinckii NCIMB 8052. An in silico screen of the C. beijerinckii genome revealed eight potential acetoin reductases. One of them (CBEI_1464) showed substantial acetoin reductase activity after expression in Escherichia coli. The purified enzyme (C. beijerinckii acetoin reductase [Cb-ACR]) was found to exist predominantly as a homodimer. In addition to acetoin (or 2,3-butanediol), other secondary alcohols and corresponding ketones were converted as well, provided that another electronegative group was attached to the adjacent C-3 carbon. Optimal activity was at pH 6.5 (reduction) and 9.5 (oxidation) and around 68°C. Cb-ACR accepts both NADH and NADPH as electron donors; however, unlike closely related enzymes, NADPH is preferred (Km, 32 µM). Cb-ACR was compared to characterized close homologs, all belonging to the "threonine dehydrogenase and related Zn-dependent dehydrogenases" (COG1063). Metal analysis confirmed the presence of 2 Zn(2+) atoms. To gain insight into the substrate and cofactor specificity, a structural model was constructed. The catalytic zinc atom is likely coordinated by Cys37, His70, and Glu71, while the structural zinc site is probably composed of Cys100, Cys103, Cys106, and Cys114. Residues determining NADP specificity were predicted as well. The physiological role of Cb-ACR in C. beijerinckii is discussed

KW - l-threonine dehydrogenase

KW - protein-structure prediction

KW - crystal-structure

KW - alcohol dehydrogenases

KW - (2r,3r)-2,3-butanediol dehydrogenase

KW - thermoanaerobacter-brockii

KW - escherichia-coli

KW - meso-2,3-butanediol dehydrogenase

KW - sulfolobus-solfataricus

KW - butanol fer

U2 - 10.1128/AEM.04007-13

DO - 10.1128/AEM.04007-13

M3 - Article

VL - 80

SP - 2011

EP - 2020

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 6

ER -