Structural Basis for a Kolbe-Type Decarboxylation Catalyzed by a Glycyl Radical Enzyme

Abstract

4-Hydroxyphenylacetate decarboxylase is a [4Fe-4S] cluster containing glycyl radical enzyme proposed to use a glycyl/thiyl radical dyad to catalyze the last step of tyrosine fermentation in clostridia. The decarboxylation product p-cresol (4-methylphenol) is a virulence factor of the human pathogen Clostridium difficile . Here we describe the crystal structures at 1.75 and 1.81 Å resolution of substrate-free and substrate-bound 4-hydroxyphenylacetate decarboxylase from the related Clostridium scatologenes . The structures show a (βγ)(4) tetramer of heterodimers composed of a catalytic β-subunit harboring the putative glycyl/thiyl dyad and a distinct small γ-subunit with two [4Fe-4S] clusters at 40 Å distance from the active site. The γ-subunit comprises two domains displaying pseudo-2-fold symmetry that are structurally related to the [4Fe-4S] cluster-binding scaffold of high-potential iron-sulfur proteins. The N-terminal domain coordinates one cluster with one histidine and three cysteines, and the C-terminal domain coordinates the second cluster with four cysteines. Whereas the C-terminal cluster is buried in the βγ heterodimer interface, the N-terminal cluster is not part of the interface. The previously postulated decarboxylation mechanism required the substrate's hydroxyl group in the vicinity of the active cysteine residue. In contrast to expectation, the substrate-bound state shows a direct interaction between the substrate's carboxyl group and the active site Cys503, while His536 and Glu637 at the opposite side of the active site pocket anchor the hydroxyl group. This state captures a possible catalytically competent complex and suggests a Kolbe-type decarboxylation for p-cresol formation.

1,2-Propanediol Dehydration in Roseburia inulinivorans

“…This is not surprising considering that maintaining the glycyl radical during crystal growth is simply not feasible. Despite this technical detail, the crystal structures for GREs have provided significant insight into enzyme mechanism (3,(22)(23)(24)(25)(26)(27)(28). Numerous computational predictions have also been made based on this structural information (29 -31).…”

“…Recent work with CutC has provided evidence that the substrate itself may help induce some of the conformational changes required for activation (15). In addition to the CbGD, the overall fold of the RiDD is also more similar to the large subunits of benzyl succinate synthase and 4-hydroxyphenyl acetate decarboxylase (24,40). However, both benzyl succinate synthase and 4-hydroxyphenyl acetate decarboxylase have additional peptide subunits, presenting an additional challenge when moving the glycyl radical loop out of the C-terminal domain and into the active site of their respective activating enzymes (25).…”

Diversity of Benzylsuccinate Synthase-Like ( bssA ) Genes in Hydrocarbon-Polluted Marine Sediments Suggests Substrate-Dependent Clustering

“…The sequences in the amplified PCR fragments analyzed in this work included the active site of the enzyme, especially the conserved Cys residue directly involved in the abstraction of a hydrogen atom from the substrate (10). The binding pocket of the active site has been accurately defined by the structure of four crystallized proteins belonging to the radical enzyme family: Clostridium butyricum glycerol dehydratase (59), Archaeoglobus fulgidus PFL2 (60), Clostridium scatologenes hydroxyphenylacetate decarboxylase (61), and Escherichia coli pyruvate formatelyase (62). The residue positions shaping this pocket have been identified in each structure and mapped in the multiple-sequence alignment.…”

Structures of benzylsuccinate synthase elucidate roles of accessory subunits in glycyl radical enzyme activation and activity

“…These atypical redox properties support the consideration of redox roles as well as structural roles for the clusters of BSS. For HPAD, it has been proposed that the [4Fe-4S] clusters are redox active and could act to reductively quench the Gly radical by electron transfer, returning the enzyme to the inactive state (18). Efficient, reversible reduction of the Gly radical has been observed in vitro in PFL in the presence of DTT or β-mercaptoethanol (29), suggesting that Gly radical reduction could be a means of preventing permanent damage to the GRE by quenching the radical before O 2 -catalyzed backbone cleavage can occur.…”

“…The only structural information available for multisubunit GREs comes from HPAD. The structure of the catalytic subunit of HPAD, HpdB, in complex with its small subunit, HpdC, was recently solved, revealing that HpdC is bound at the protein's surface away from the active site and the Gly radical domain (18).…”