Development of an in vitro method for activation of X-succinate synthases for fumarate hydroalkylation
Summary: Anaerobic microbial degradation of hydrocarbons is often initiated through addition of the hydrocarbon to fumarate by enzymes known as X-succinate synthases (XSSs). XSSs use a glycyl radical cofactor, which is installed by an activating enzyme (XSS-AE), to catalyze this carbon-carbon coupli...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2023-06-01
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| Series: | iScience |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2589004223009793 |
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| author | Mary C. Andorfer Devin T. King-Roberts Christa N. Imrich Balyn G. Brotheridge Catherine L. Drennan |
| author_facet | Mary C. Andorfer Devin T. King-Roberts Christa N. Imrich Balyn G. Brotheridge Catherine L. Drennan |
| author_sort | Mary C. Andorfer |
| collection | DOAJ |
| description | Summary: Anaerobic microbial degradation of hydrocarbons is often initiated through addition of the hydrocarbon to fumarate by enzymes known as X-succinate synthases (XSSs). XSSs use a glycyl radical cofactor, which is installed by an activating enzyme (XSS-AE), to catalyze this carbon-carbon coupling reaction. The activation step, although crucial for catalysis, has not previously been possible in vitro because of insolubility of XSS-AEs. Here, we take a genome mining approach to find an XSS-AE, a 4-isopropylbenzylsuccinate synthase (IBSS)-AE (IbsAE) that can be solubly expressed in Escherichia coli. This soluble XSS-AE can activate both IBSS and the well-studied benzylsuccinate synthase (BSS) in vitro, allowing us to explore XSSs biochemically. To start, we examine the role of BSS subunits and find that the beta subunit accelerates the rate of hydrocarbon addition. Looking forward, the methodology and insight gathered here can be used more broadly to understand and engineer XSSs as synthetically useful biocatalysts. |
| first_indexed | 2024-03-13T08:06:24Z |
| format | Article |
| id | doaj.art-7f0ec84e99054e999b06e030f6a72697 |
| institution | Directory Open Access Journal |
| issn | 2589-0042 |
| language | English |
| last_indexed | 2024-03-13T08:06:24Z |
| publishDate | 2023-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | iScience |
| spelling | doaj.art-7f0ec84e99054e999b06e030f6a726972023-06-01T04:36:47ZengElsevieriScience2589-00422023-06-01266106902Development of an in vitro method for activation of X-succinate synthases for fumarate hydroalkylationMary C. Andorfer0Devin T. King-Roberts1Christa N. Imrich2Balyn G. Brotheridge3Catherine L. Drennan4Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USADepartment of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USADepartment of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USADepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USADepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Center for Environmental Health, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Bio-inspired Solar Energy Program, Canadian Institute for Advanced Research (CIFAR), Toronto, ON M5G 1M1, Canada; Corresponding authorSummary: Anaerobic microbial degradation of hydrocarbons is often initiated through addition of the hydrocarbon to fumarate by enzymes known as X-succinate synthases (XSSs). XSSs use a glycyl radical cofactor, which is installed by an activating enzyme (XSS-AE), to catalyze this carbon-carbon coupling reaction. The activation step, although crucial for catalysis, has not previously been possible in vitro because of insolubility of XSS-AEs. Here, we take a genome mining approach to find an XSS-AE, a 4-isopropylbenzylsuccinate synthase (IBSS)-AE (IbsAE) that can be solubly expressed in Escherichia coli. This soluble XSS-AE can activate both IBSS and the well-studied benzylsuccinate synthase (BSS) in vitro, allowing us to explore XSSs biochemically. To start, we examine the role of BSS subunits and find that the beta subunit accelerates the rate of hydrocarbon addition. Looking forward, the methodology and insight gathered here can be used more broadly to understand and engineer XSSs as synthetically useful biocatalysts.http://www.sciencedirect.com/science/article/pii/S2589004223009793BiochemistryBiocatalysisStructural biologyBioengineering |
| spellingShingle | Mary C. Andorfer Devin T. King-Roberts Christa N. Imrich Balyn G. Brotheridge Catherine L. Drennan Development of an in vitro method for activation of X-succinate synthases for fumarate hydroalkylation iScience Biochemistry Biocatalysis Structural biology Bioengineering |
| title | Development of an in vitro method for activation of X-succinate synthases for fumarate hydroalkylation |
| title_full | Development of an in vitro method for activation of X-succinate synthases for fumarate hydroalkylation |
| title_fullStr | Development of an in vitro method for activation of X-succinate synthases for fumarate hydroalkylation |
| title_full_unstemmed | Development of an in vitro method for activation of X-succinate synthases for fumarate hydroalkylation |
| title_short | Development of an in vitro method for activation of X-succinate synthases for fumarate hydroalkylation |
| title_sort | development of an in vitro method for activation of x succinate synthases for fumarate hydroalkylation |
| topic | Biochemistry Biocatalysis Structural biology Bioengineering |
| url | http://www.sciencedirect.com/science/article/pii/S2589004223009793 |
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