PMID- 37283811
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20230726
IS  - 2589-0042 (Electronic)
IS  - 2589-0042 (Linking)
VI  - 26
IP  - 6
DP  - 2023 Jun 16
TI  - Development of an in vitro method for activation of X-succinate synthases for 
      fumarate hydroalkylation.
PG  - 106902
LID - 10.1016/j.isci.2023.106902 [doi]
LID - 106902
AB  - 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.
CI  - (c) 2023 The Author(s).
FAU - Andorfer, Mary C
AU  - Andorfer MC
AD  - Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 
      02139, USA.
AD  - Howard Hughes Medical Institute, Massachusetts Institute of Technology, 
      Cambridge, MA 02139, USA.
FAU - King-Roberts, Devin T
AU  - King-Roberts DT
AD  - Department of Biological Engineering, Massachusetts Institute of Technology, 
      Cambridge, MA 02139, USA.
FAU - Imrich, Christa N
AU  - Imrich CN
AD  - Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 
      02139, USA.
FAU - Brotheridge, Balyn G
AU  - Brotheridge BG
AD  - Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 
      02139, USA.
AD  - Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 
      02139, USA.
FAU - Drennan, Catherine L
AU  - Drennan CL
AD  - Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 
      02139, USA.
AD  - Howard Hughes Medical Institute, Massachusetts Institute of Technology, 
      Cambridge, MA 02139, USA.
AD  - Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 
      02139, USA.
AD  - Center for Environmental Health, Massachusetts Institute of Technology, 
      Cambridge, MA 02139, USA.
AD  - Bio-inspired Solar Energy Program, Canadian Institute for Advanced Research 
      (CIFAR), Toronto, ON M5G 1M1, Canada.
LA  - eng
GR  - F32 GM129882/GM/NIGMS NIH HHS/United States
GR  - K99 GM145910/GM/NIGMS NIH HHS/United States
GR  - P30 ES002109/ES/NIEHS NIH HHS/United States
GR  - R35 GM126982/GM/NIGMS NIH HHS/United States
PT  - Journal Article
DEP - 20230519
PL  - United States
TA  - iScience
JT  - iScience
JID - 101724038
PMC - PMC10239695
OTO - NOTNLM
OT  - Biocatalysis
OT  - Biochemistry
OT  - Bioengineering
OT  - Structural biology
COIS- The authors declare no competing interests.
EDAT- 2023/06/07 13:10
MHDA- 2023/06/07 13:11
PMCR- 2023/05/19
CRDT- 2023/06/07 09:38
PHST- 2023/04/19 00:00 [received]
PHST- 2023/05/08 00:00 [revised]
PHST- 2023/05/12 00:00 [accepted]
PHST- 2023/06/07 13:11 [medline]
PHST- 2023/06/07 13:10 [pubmed]
PHST- 2023/06/07 09:38 [entrez]
PHST- 2023/05/19 00:00 [pmc-release]
AID - S2589-0042(23)00979-3 [pii]
AID - 106902 [pii]
AID - 10.1016/j.isci.2023.106902 [doi]
PST - epublish
SO  - iScience. 2023 May 19;26(6):106902. doi: 10.1016/j.isci.2023.106902. eCollection 
      2023 Jun 16.