Catalytic performance of a class III Old yellow enzyme and its cysteine variants

Anika Scholtissek, Eric Gädke, Caroline E. Paul, Adrie H. Westphal, Willem J.H. van Berkel, Dirk Tischler*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

4 Citations (Scopus)

Abstract

Class III old yellow enzymes (OYEs) contain a conserved cysteine in their active sites. To address the role of this cysteine in OYE-mediated asymmetric synthesis, we have studied the biocatalytic properties of OYERo2a from Rhodococcus opacus 1CP (WT) as well as its engineered variants C25A, C25S and C25G. OYERo2a in its redox resting state (oxidized form) is irreversibly inactivated by N-methylmaleimide. As anticipated, inactivation does not occur with the Cys variants. Steady-state kinetics with this maleimide substrate revealed that C25S and C25G doubled the turnover frequency (kcat) while showing increased KM values compared to WT, and that C25A performed more similar to WT. Applying the substrate 2-cyclohexen-1-one, the Cys variants were less active and less efficient than WT. OYERo2a and its Cys variants showed different activities with NADPH, the natural reductant. The variants did bind NADPH less well but kcat was significantly increased. The most efficient variant was C25G. Replacement of NADPH with the cost-effective synthetic cofactor 1-benzyl-1,4-dihydronicotinamide (BNAH) drastically changed the catalytic behavior. Again C25G was most active and showed a similar efficiency as WT. Biocatalysis experiments showed that OYERo2a, C25S, and C25G converted N-phenyl-2-methylmaleimide equally well (81-84%) with an enantiomeric excess (ee) of more than 99% for the R-product. With cyclic ketones, the highest conversion (89%) and ee (>99%) was observed for the reaction of WT with R-carvone. A remarkable poor conversion of cyclic ketones occurred with C25G. In summary, we established that the generation of a cysteine-free enzyme and cofactor optimization allows the development of more robust class III OYEs.

Original languageEnglish
Article number02410
JournalFrontiers in Microbiology
Volume9
Issue numberOCT
DOIs
Publication statusPublished - 12 Oct 2018

Fingerprint

NADPH Dehydrogenase
NADP
Cysteine
Ketones
Biocatalysis
Rhodococcus
Coenzymes
Reducing Agents
Oxidation-Reduction
Catalytic Domain
Costs and Cost Analysis

Keywords

  • Actinobacteria
  • Biocatalysis
  • Cysteine modification
  • Ene reductase
  • Flavoprotein
  • Inactivation
  • Protein engineering
  • Rhodococcus opacus 1CP

Cite this

Scholtissek, Anika ; Gädke, Eric ; Paul, Caroline E. ; Westphal, Adrie H. ; van Berkel, Willem J.H. ; Tischler, Dirk. / Catalytic performance of a class III Old yellow enzyme and its cysteine variants. In: Frontiers in Microbiology. 2018 ; Vol. 9, No. OCT.
@article{3b2631321c784804b5903595aeee0a31,
title = "Catalytic performance of a class III Old yellow enzyme and its cysteine variants",
abstract = "Class III old yellow enzymes (OYEs) contain a conserved cysteine in their active sites. To address the role of this cysteine in OYE-mediated asymmetric synthesis, we have studied the biocatalytic properties of OYERo2a from Rhodococcus opacus 1CP (WT) as well as its engineered variants C25A, C25S and C25G. OYERo2a in its redox resting state (oxidized form) is irreversibly inactivated by N-methylmaleimide. As anticipated, inactivation does not occur with the Cys variants. Steady-state kinetics with this maleimide substrate revealed that C25S and C25G doubled the turnover frequency (kcat) while showing increased KM values compared to WT, and that C25A performed more similar to WT. Applying the substrate 2-cyclohexen-1-one, the Cys variants were less active and less efficient than WT. OYERo2a and its Cys variants showed different activities with NADPH, the natural reductant. The variants did bind NADPH less well but kcat was significantly increased. The most efficient variant was C25G. Replacement of NADPH with the cost-effective synthetic cofactor 1-benzyl-1,4-dihydronicotinamide (BNAH) drastically changed the catalytic behavior. Again C25G was most active and showed a similar efficiency as WT. Biocatalysis experiments showed that OYERo2a, C25S, and C25G converted N-phenyl-2-methylmaleimide equally well (81-84{\%}) with an enantiomeric excess (ee) of more than 99{\%} for the R-product. With cyclic ketones, the highest conversion (89{\%}) and ee (>99{\%}) was observed for the reaction of WT with R-carvone. A remarkable poor conversion of cyclic ketones occurred with C25G. In summary, we established that the generation of a cysteine-free enzyme and cofactor optimization allows the development of more robust class III OYEs.",
keywords = "Actinobacteria, Biocatalysis, Cysteine modification, Ene reductase, Flavoprotein, Inactivation, Protein engineering, Rhodococcus opacus 1CP",
author = "Anika Scholtissek and Eric G{\"a}dke and Paul, {Caroline E.} and Westphal, {Adrie H.} and {van Berkel}, {Willem J.H.} and Dirk Tischler",
year = "2018",
month = "10",
day = "12",
doi = "10.3389/fmicb.2018.02410",
language = "English",
volume = "9",
journal = "Frontiers in Microbiology",
issn = "1664-302X",
publisher = "Frontiers",
number = "OCT",

}

Catalytic performance of a class III Old yellow enzyme and its cysteine variants. / Scholtissek, Anika; Gädke, Eric; Paul, Caroline E.; Westphal, Adrie H.; van Berkel, Willem J.H.; Tischler, Dirk.

In: Frontiers in Microbiology, Vol. 9, No. OCT, 02410, 12.10.2018.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Catalytic performance of a class III Old yellow enzyme and its cysteine variants

AU - Scholtissek, Anika

AU - Gädke, Eric

AU - Paul, Caroline E.

AU - Westphal, Adrie H.

AU - van Berkel, Willem J.H.

AU - Tischler, Dirk

PY - 2018/10/12

Y1 - 2018/10/12

N2 - Class III old yellow enzymes (OYEs) contain a conserved cysteine in their active sites. To address the role of this cysteine in OYE-mediated asymmetric synthesis, we have studied the biocatalytic properties of OYERo2a from Rhodococcus opacus 1CP (WT) as well as its engineered variants C25A, C25S and C25G. OYERo2a in its redox resting state (oxidized form) is irreversibly inactivated by N-methylmaleimide. As anticipated, inactivation does not occur with the Cys variants. Steady-state kinetics with this maleimide substrate revealed that C25S and C25G doubled the turnover frequency (kcat) while showing increased KM values compared to WT, and that C25A performed more similar to WT. Applying the substrate 2-cyclohexen-1-one, the Cys variants were less active and less efficient than WT. OYERo2a and its Cys variants showed different activities with NADPH, the natural reductant. The variants did bind NADPH less well but kcat was significantly increased. The most efficient variant was C25G. Replacement of NADPH with the cost-effective synthetic cofactor 1-benzyl-1,4-dihydronicotinamide (BNAH) drastically changed the catalytic behavior. Again C25G was most active and showed a similar efficiency as WT. Biocatalysis experiments showed that OYERo2a, C25S, and C25G converted N-phenyl-2-methylmaleimide equally well (81-84%) with an enantiomeric excess (ee) of more than 99% for the R-product. With cyclic ketones, the highest conversion (89%) and ee (>99%) was observed for the reaction of WT with R-carvone. A remarkable poor conversion of cyclic ketones occurred with C25G. In summary, we established that the generation of a cysteine-free enzyme and cofactor optimization allows the development of more robust class III OYEs.

AB - Class III old yellow enzymes (OYEs) contain a conserved cysteine in their active sites. To address the role of this cysteine in OYE-mediated asymmetric synthesis, we have studied the biocatalytic properties of OYERo2a from Rhodococcus opacus 1CP (WT) as well as its engineered variants C25A, C25S and C25G. OYERo2a in its redox resting state (oxidized form) is irreversibly inactivated by N-methylmaleimide. As anticipated, inactivation does not occur with the Cys variants. Steady-state kinetics with this maleimide substrate revealed that C25S and C25G doubled the turnover frequency (kcat) while showing increased KM values compared to WT, and that C25A performed more similar to WT. Applying the substrate 2-cyclohexen-1-one, the Cys variants were less active and less efficient than WT. OYERo2a and its Cys variants showed different activities with NADPH, the natural reductant. The variants did bind NADPH less well but kcat was significantly increased. The most efficient variant was C25G. Replacement of NADPH with the cost-effective synthetic cofactor 1-benzyl-1,4-dihydronicotinamide (BNAH) drastically changed the catalytic behavior. Again C25G was most active and showed a similar efficiency as WT. Biocatalysis experiments showed that OYERo2a, C25S, and C25G converted N-phenyl-2-methylmaleimide equally well (81-84%) with an enantiomeric excess (ee) of more than 99% for the R-product. With cyclic ketones, the highest conversion (89%) and ee (>99%) was observed for the reaction of WT with R-carvone. A remarkable poor conversion of cyclic ketones occurred with C25G. In summary, we established that the generation of a cysteine-free enzyme and cofactor optimization allows the development of more robust class III OYEs.

KW - Actinobacteria

KW - Biocatalysis

KW - Cysteine modification

KW - Ene reductase

KW - Flavoprotein

KW - Inactivation

KW - Protein engineering

KW - Rhodococcus opacus 1CP

U2 - 10.3389/fmicb.2018.02410

DO - 10.3389/fmicb.2018.02410

M3 - Article

VL - 9

JO - Frontiers in Microbiology

JF - Frontiers in Microbiology

SN - 1664-302X

IS - OCT

M1 - 02410

ER -