Structural basis for anaerobic alkane activation by a multisubunit glycyl radical enzyme

• 成果类型: Article
• 署名作者: Andorfer, Mary C.; Levitz, Talya S.; Liu, Jian; Chakraborty, Ankush; King-Roberts, Devin T.; Nweneka, Delight; Imrich, Christa N.; Drennan, Catherine L.
• 署名单位: Michigan State University; Massachusetts Institute of Technology (MIT); Howard Hughes Medical Institute; Massachusetts Institute of Technology (MIT); Massachusetts Institute of Technology (MIT); Massachusetts Institute of Technology (MIT)
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-10080
• DOI: 10.1073/pnas.2510389122
• 发表日期: 2025-08-12
• 关键词: p-hydroxyphenylacetate decarboxylase; benzylsuccinate synthase; thauera-aromatica; denitrifying bacterium; n-alkanes; cryo-em; toluene; oxidation; hydrocarbons; mechanism

摘要

X-succinate synthases (XSSs) are glycyl radical enzymes (GREs) that catalyze the addition of hydrocarbons to fumarate via radical chemistry, thereby activating them for microbial metabolism. To date, the only structurally characterized XSS is benzylsuccinate synthase (BSS), which functionalizes toluene. A distinct subclass ofXSSs acts on saturated hydrocarbons, which possess much stronger C(sp3)-H bonds than toluene, suggesting mechanistic and structural differences from BSS. Here, we use cryogenic electron microscopy to determine the structure of one such enzyme, (1-methylalkyl)succinate synthase (MASS) from Azoarcus strain HxN1, which functionalizes n-alkanes (C6-C8). The structure reveals an asymmetric dimer in which both sides contain a catalytic alpha- subunit and accessory gamma-subunit. One alpha- subunit also binds two additional subunits, beta and delta. The beta- subunit binds a [4Fe-4S] cluster and adopts a fold similar to BSS beta. The beta- subunit appears to regulate the flexibility of the alpha- subunit to enable opening of the active site, affording the binding of n-alkane substrates. The delta- subunit, which lacks homology to known GRE subunits, adopts a rubredoxin-like fold that binds a single Fe ion, an architecture not previously reported for GREs. MASS delta occupies the same region of the alpha- subunit as the activating enzyme (AE) and may regulate the conformational changes required for glycyl radical installation. Structural comparisons between MASS and BSS reveal differences in how fumarate is bound and show amino acid substitutions that could account for the binding of alkanes versus toluene. Together, this structure offers insight into anaerobic alkane activation via fumarate addition.