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

ABSTRACT: Anaerobic degradation of the environmental pollutant toluene is initiated by the glycyl radical enzyme benzylsuccinate synthase (BSS), which catalyzes the radical addition of toluene to fumarate, forming benzylsuccinate. We have determined crystal structures of the catalytic ?-subunit of BSS with its accessory subunits ? and ?, which both bind a [4Fe-4S] cluster and are essential for BSS activity in vivo. We find that BSS? has the common glycyl radical enzyme fold, a 10-stranded ?/?-barrel that surrounds the glycyl radical cofactor and active site. Both accessory subunits ? and ? display folds related to high potential iron-sulfur proteins but differ substantially from each other in how they interact with the ?-subunit. BSS? binds distally to the active site, burying a hydrophobic region of BSS?, whereas BSS? binds to a hydrophilic surface of BSS? that is proximal to the active site. To further investigate the function of BSS?, we determined the structure of a BSS?? complex. Remarkably, we find that the barrel partially opens, allowing the C-terminal region of BSS? that houses the glycyl radical to shift within the barrel toward an exit pathway. The structural changes that we observe in the BSS?? complex center around the crucial glycyl radical domain, thus suggesting a role for BSS? in modulating the conformational dynamics required for enzyme activity. Accompanying proteolysis experiments support these structural observations.

SUBMITTER: Funk MA

PROVIDER: S-EPMC4104874 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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

Funk Michael A MA Judd Evan T ET Marsh E Neil G EN Elliott Sean J SJ Drennan Catherine L CL

Proceedings of the National Academy of Sciences of the United States of America 20140630 28

Anaerobic degradation of the environmental pollutant toluene is initiated by the glycyl radical enzyme benzylsuccinate synthase (BSS), which catalyzes the radical addition of toluene to fumarate, forming benzylsuccinate. We have determined crystal structures of the catalytic α-subunit of BSS with its accessory subunits β and γ, which both bind a [4Fe-4S] cluster and are essential for BSS activity in vivo. We find that BSSα has the common glycyl radical enzyme fold, a 10-stranded β/α-barrel that ...[more]

PMID: 24982148

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Project description:Bacterial microcompartments (BMCs) are large (∼100-nm) protein shells that encapsulate enzymes, their substrates, and cofactors for the purposes of increasing metabolic reaction efficiency and protecting cells from toxic intermediates. The best-studied microcompartment is the carbon-fixing carboxysome that encapsulates ribulose-1,5-bisphosphate carboxylase and carbonic anhydrase. Other well-known BMCs include the Pdu and Eut BMCs, which metabolize 1,2-propanediol and ethanolamine, respectively, with vitamin B12-dependent diol dehydratase enzymes. Recent bioinformatic analyses identified a new prevalent type of BMC, hypothesized to utilize vitamin B12-independent glycyl radical enzymes to metabolize substrates. Here we use genetic and metabolic analyses to undertake in vivo characterization of the newly identified glycyl radical enzyme microcompartment 3 (GRM3) class of microcompartment clusters. Transcriptome sequencing analyses showed that the microcompartment gene cluster in the genome of the purple photosynthetic bacterium Rhodobacter capsulatus was expressed under dark anaerobic respiratory conditions in the presence of 1,2-propanediol. High-performance liquid chromatography and gas chromatography-mass spectrometry analyses showed that enzymes coded by this cluster metabolized 1,2-propanediol into propionaldehyde, propanol, and propionate. Surprisingly, the microcompartment pathway did not protect these cells from toxic propionaldehyde under the conditions used in this study, with buildup of this intermediate contributing to arrest of cell growth. We further show that expression of microcompartment genes is regulated by a two-component system located downstream of the microcompartment cluster.IMPORTANCE BMCs are protein shells that are designed to compartmentalize enzymatic reactions that require either sequestration of a substrate or the sequestration of toxic intermediates. Due to their ability to compartmentalize reactions, BMCs have also become attractive targets for bioengineering novel enzymatic reactions. Despite these useful features, little is known about the biochemistry of newly identified classes of BMCs. In this study, we have undertaken genetic and in vivo metabolic analyses of the newly identified GRM3 gene cluster.

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

Publications

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

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