Development of an in vitro method for activation of X-succinate synthases for fumarate hydroalkylation
Mary C. Andorfer,
Devin T. King-Roberts,
Christa N. Imrich,
Balyn G. Brotheridge,
Catherine L. Drennan
Affiliations
Mary C. Andorfer
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Devin T. King-Roberts
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Christa N. Imrich
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Balyn G. Brotheridge
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Catherine L. Drennan
Department 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 author
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.
