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Benzylsuccinate synthase alpha-beta-gamma complex with bound toluene and fumarate
Structural highlights
FunctionPublication Abstract from PubMedX-succinate synthases (XSSs) are a class of glycyl radical enzymes (GREs) that enable anaerobic hydrocarbon functionalization, granting anaerobes access to petroleum-derived substrates for metabolism. Owing to their ability to functionalize components of crude oil and catalyze selective olefin hydroalkylation, XSSs hold significant biotechnological promise. However, mechanistic understanding has been limited due to long-standing barriers to installing their essential glycyl radical in vitro, which have only recently been overcome. Unlike most GREs, XSSs contain accessory subunits that bind to the periphery of the catalytic subunit. The most well-studied XSS, benzylsuccinate synthase (BSS), includes two [4Fe-4S] cluster-binding accessory subunits, BSSgamma and BSSbeta. The full structure of BSSgamma and the catalytic role of BSSbeta have remained unclear. Here, we report the crystal structure of BSSgamma with its [4Fe-4S] cluster intact, revealing a HiPP-like fold similar to that of BSSbeta. Through biochemical and spectroscopic studies, we provide evidence that BSSbeta promotes thiyl radical formation, even in the absence of a substrate. This finding contrasts with recent models, in which substrate binding is required to trigger thiyl radical formation. With this mechanistic insight, we optimized reaction conditions to achieve total turnover numbers of approximately 17,000, representing an over 340-fold improvement compared to prior reports. We further show that in the absence of BSSbeta, activated BSSalphagamma remains catalytically active for up to 11 days. Together, these results clarify the unique regulatory architecture of BSS and lay the groundwork for the use of XSSs in biocatalytic applications. Accessory Subunit Regulates Thiyl Radical Formation in Benzylsuccinate Synthase.,Anas S, Liu J, Vats A, Gainadi R, Sharif S, Piriyatamwong A, Andorfer MC Biochemistry. 2025 Oct 13. doi: 10.1021/acs.biochem.5c00492. PMID:41077954[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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