Details
| Original language | English |
|---|---|
| Pages (from-to) | 4044-4051 |
| Number of pages | 8 |
| Journal | CHEMCATCHEM |
| Volume | 12 |
| Issue number | 16 |
| Early online date | 28 Jul 2020 |
| Publication status | Published - 21 Aug 2020 |
| Externally published | Yes |
Abstract
Biocatalysis is increasingly used in combination with light to develop new and more sustainable synthetic methods. Thereby, mostly a chemical photocatalyst harvesting the light energy is combined with an established enzymatic reaction, thus the biocatalyst itself does not require the light for its specific reaction. Here we expand the library of an enzyme which requires light for its natural reaction, namely the light-dependent protochlorophyllide oxidoreductase (LPOR). This enzyme catalyzes the NADPH-dependent reduction of a C=C in a N-heterocycle. Out of five LPORs identified by sequence search, four were found to be well expressible in E. coli and active. Investigating the light intensity, which is an important parameter describing energy input and subsequently may enable fast reaction, it turned out that the four LPORs can stand the maximum light intensity reachable with the equipment used (1450 μmol photons m−2 s−1). However, the natural substrate and product were degraded at these conditions, allowing only 15 % of the maximum input (211 μmol photons m−2 s−1). Furthermore, the LPORs accepted seven different water miscible solvents with a solvent content of up to 20 % v/v and were active at a pH from 6 to 10. While all LPORs known to date are exclusively NADPH dependent, two LPORs identified here were active also with NADH. The cofactor selectivity could be pinned to three amino acid residues, which interestingly do not directly bind to the cofactor.
Keywords
- Biocatalysis, biotransformations, C=C reduction, photocatalysis, photoenzymes
ASJC Scopus subject areas
- Chemical Engineering(all)
- Catalysis
- Chemistry(all)
- Physical and Theoretical Chemistry
- Chemistry(all)
- Organic Chemistry
- Chemistry(all)
- Inorganic Chemistry
Cite this
- Standard
- Harvard
- Apa
- Vancouver
- BibTeX
- RIS
In: CHEMCATCHEM, Vol. 12, No. 16, 21.08.2020, p. 4044-4051.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Extending the Library of Light-Dependent Protochlorophyllide Oxidoreductases and their Solvent Tolerance, Stability in Light and Cofactor Flexibility
AU - Schmermund, Luca
AU - Bierbaumer, Sarah
AU - Schein, Viktor K.
AU - Winkler, Christoph K.
AU - Kara, Selin
AU - Kroutil, Wolfgang
N1 - Funding Information: This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sk?odowska-Curie grant agreement No 764920. S.B. received funding from the Austrian Science Fund (FWF): DOC 46-821. We thank Dr. Derren Heyes, University of Manchester, Institute of Biotechnology, for providing the Rhodobacter capsulatus ZY5 strain and Dr. Thomas Drepper and Dr. Ulrich Krauss, Heinrich-Heine-University D?sseldorf, research center J?lich, Institute of Molecular Enzyme Technology, for providing four LPOR plasmids: pet28a-his6-Hordeum vulgare-lpor, pet28a-arabidopsis thaliana lpor-his6, pet28a-his6-thermosynechococcus elongatus-lpor and pet28a-his6-dinoroseobacter shibae-lpor. The University of Graz and the Field of Excellence BioHealth are acknowledged for financial support.
PY - 2020/8/21
Y1 - 2020/8/21
N2 - Biocatalysis is increasingly used in combination with light to develop new and more sustainable synthetic methods. Thereby, mostly a chemical photocatalyst harvesting the light energy is combined with an established enzymatic reaction, thus the biocatalyst itself does not require the light for its specific reaction. Here we expand the library of an enzyme which requires light for its natural reaction, namely the light-dependent protochlorophyllide oxidoreductase (LPOR). This enzyme catalyzes the NADPH-dependent reduction of a C=C in a N-heterocycle. Out of five LPORs identified by sequence search, four were found to be well expressible in E. coli and active. Investigating the light intensity, which is an important parameter describing energy input and subsequently may enable fast reaction, it turned out that the four LPORs can stand the maximum light intensity reachable with the equipment used (1450 μmol photons m−2 s−1). However, the natural substrate and product were degraded at these conditions, allowing only 15 % of the maximum input (211 μmol photons m−2 s−1). Furthermore, the LPORs accepted seven different water miscible solvents with a solvent content of up to 20 % v/v and were active at a pH from 6 to 10. While all LPORs known to date are exclusively NADPH dependent, two LPORs identified here were active also with NADH. The cofactor selectivity could be pinned to three amino acid residues, which interestingly do not directly bind to the cofactor.
AB - Biocatalysis is increasingly used in combination with light to develop new and more sustainable synthetic methods. Thereby, mostly a chemical photocatalyst harvesting the light energy is combined with an established enzymatic reaction, thus the biocatalyst itself does not require the light for its specific reaction. Here we expand the library of an enzyme which requires light for its natural reaction, namely the light-dependent protochlorophyllide oxidoreductase (LPOR). This enzyme catalyzes the NADPH-dependent reduction of a C=C in a N-heterocycle. Out of five LPORs identified by sequence search, four were found to be well expressible in E. coli and active. Investigating the light intensity, which is an important parameter describing energy input and subsequently may enable fast reaction, it turned out that the four LPORs can stand the maximum light intensity reachable with the equipment used (1450 μmol photons m−2 s−1). However, the natural substrate and product were degraded at these conditions, allowing only 15 % of the maximum input (211 μmol photons m−2 s−1). Furthermore, the LPORs accepted seven different water miscible solvents with a solvent content of up to 20 % v/v and were active at a pH from 6 to 10. While all LPORs known to date are exclusively NADPH dependent, two LPORs identified here were active also with NADH. The cofactor selectivity could be pinned to three amino acid residues, which interestingly do not directly bind to the cofactor.
KW - Biocatalysis
KW - biotransformations
KW - C=C reduction
KW - photocatalysis
KW - photoenzymes
UR - http://www.scopus.com/inward/record.url?scp=85088575637&partnerID=8YFLogxK
U2 - 10.1002/cctc.202000561
DO - 10.1002/cctc.202000561
M3 - Article
AN - SCOPUS:85088575637
VL - 12
SP - 4044
EP - 4051
JO - CHEMCATCHEM
JF - CHEMCATCHEM
SN - 1867-3880
IS - 16
ER -