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Development of an in vitro method for activation of X-succinate synthases for fumarate hydroalkylation

Authors :
Mary C. Andorfer
Devin T. King-Roberts
Christa N. Imrich
Balyn G. Brotheridge
Catherine L. Drennan
Source :
iScience, Vol 26, Iss 6, Pp 106902- (2023)
Publication Year :
2023
Publisher :
Elsevier, 2023.

Abstract

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.

Details

Language :
English
ISSN :
25890042
Volume :
26
Issue :
6
Database :
Directory of Open Access Journals
Journal :
iScience
Publication Type :
Academic Journal
Accession number :
edsdoj.7f0ec84e99054e999b06e030f6a72697
Document Type :
article
Full Text :
https://doi.org/10.1016/j.isci.2023.106902