Substrate-bound Structures of Benzylsuccinate Synthase Reveal How Toluene Is Activated in Anaerobic Hydrocarbon Degradation

Funk, Michael A.; Marsh, E. Neil G.; Drennan, Catherine L.

doi:10.1074/jbc.m115.670737

Cited by 40 publications

(53 citation statements)

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“…A notable feature of the CutC active site also observed in other GREs (Funk et al, 2014, Funk et al, 2015) is a hydrophobic cap formed by a helix extending from β3 (Figure 2C). This cap packs against two tyrosine residues that participate in choline binding (described below) and appears to block access to the active site from the exterior of the protein; simple rotation of the side chains of Phe389 and/or Trp379 into adjacent, solvent-filled regions may allow access to the active site.…”

Section: Resultsmentioning

confidence: 59%

Molecular Basis of C–N Bond Cleavage by the Glycyl Radical Enzyme Choline Trimethylamine-Lyase

Bodea

Funk

Balskus

et al. 2016

Cell Chemical Biology

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SUMMARY Deamination of choline catalyzed by the glycyl radical enzyme choline trimethylamine-lyase (CutC) has emerged as an important route for the production of trimethylamine, a microbial metabolite associated with both human disease and biological methane production. Here, we have determined five high-resolution X-ray structures of wild-type CutC and mechanistically informative mutants in the presence of choline. Within an unexpectedly polar active site, CutC orients choline through hydrogen bonding with a putative general base, and through close interactions between phenolic and carboxylate oxygen atoms of the protein scaffold and the polarized methyl groups of the trimethylammonium moiety. These structural data, along with biochemical analysis of active site mutants, support a mechanism that involves direct elimination of trimethylamine. This work broadens our understanding of radical-based enzyme catalysis and will aid in the rational design of inhibitors of bacterial trimethylamine production.

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Section: Resultsmentioning

confidence: 59%

Molecular Basis of C–N Bond Cleavage by the Glycyl Radical Enzyme Choline Trimethylamine-Lyase

Bodea

Funk

Balskus

et al. 2016

Cell Chemical Biology

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“…Structures of BSS show fumarate and toluene bound within the active site cavity, adopting optimal orientations for radical-mediated C-C bond formation (Funk et al , 2015) (Fig. 7C).…”

Section: Gre Ribonucleotide Reductasesmentioning

confidence: 99%

New tricks for the glycyl radical enzyme family

Backman

Funk

Dawson

et al. 2017

Critical Reviews in Biochemistry and Molecular Biology

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129

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Glycyl radical enzymes (GREs) are important biological catalysts in both strict and facultative anaerobes, playing key roles both in the human microbiota and in the environment. GREs contain a backbone glycyl radical that is post-translationally installed, enabling radical-based mechanisms. GREs function in several metabolic pathways including mixed acid fermentation, ribonucleotide reduction, and the anaerobic breakdown of the nutrient choline and the pollutant toluene. By generating a substrate-based radical species within the active site, GREs enable C-C, C-O, and C-N bond breaking and formation steps that are otherwise challenging for non-radical enzymes. Identification of previously unknown family members from genomic data and the determination of structures of well-characterized GREs have expanded the scope of GRE-catalyzed reactions as well as defined key features that enable radical catalysis. Here we review the structures and mechanisms of characterized GREs, classifying members into five categories. We consider the open questions about each of the five GRE classes and evaluate the tools available to interrogate uncharacterized GREs.

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“…1 a, upper panel). Upon substrate binding (first fumarate, then toluene in a long active site cavity of BSS from T. aromatica T1 [Funk et al, 2015]), a reactive thiyl radical (Cys 493 ) is generated at the expense of the spatially close-by resting-state glycyl radical (Gly 829 ). In the initial step controlling the reaction rate, the thiyl radical abstracts a hydrogen atom from the methyl group, yielding a first transition state ( fig.…”

Section: Metabolism Of Aromatic Compoundsmentioning

confidence: 99%

“…1 b) vated to the respective succinate derivatives (see below section 'Stereochemistry of anaerobic activation of n-alkanes and co-metabolic capacities of anaerobic n-alkane degraders'). Recent homology modeling on the basis of the BSS X-ray crystal structure [Funk et al, 2015] Heider et al [2016c]. The soluble heterotrimeric EBDH contains a Mo-bis-MGD cofactor (MoCo) in the α-subunit and is closely related to membrane-anchored nitrate reductase from Escherichia coli (NarGHI) of the DMSO reductase subfamily II.…”

Section: Metabolism Of Aromatic Compoundsmentioning

confidence: 99%

Anaerobic Microbial Degradation of Hydrocarbons: From Enzymatic Reactions to the Environment

et al. 2016

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Hydrocarbons are abundant in anoxic environments and pose biochemical challenges to their anaerobic degradation by microorganisms. Within the framework of the Priority Program 1319, investigations funded by the Deutsche Forschungsgemeinschaft on the anaerobic microbial degradation of hydrocarbons ranged from isolation and enrichment of hitherto unknown hydrocarbon-degrading anaerobic microorganisms, discovery of novel reactions, detailed studies of enzyme mechanisms and structures to process-oriented in situ studies. Selected highlights from this program are collected in this synopsis, with more detailed information provided by theme-focused reviews of the special topic issue on ‘Anaerobic biodegradation of hydrocarbons' [this issue, pp. 1-244]. The interdisciplinary character of the program, involving microbiologists, biochemists, organic chemists and environmental scientists, is best exemplified by the studies on alkyl-/arylalkylsuccinate synthases. Here, research topics ranged from in-depth mechanistic studies of archetypical toluene-activating benzylsuccinate synthase, substrate-specific phylogenetic clustering of alkyl-/arylalkylsuccinate synthases (toluene plus xylenes, p-cymene, p-cresol, 2-methylnaphthalene, n-alkanes), stereochemical and co-metabolic insights into n-alkane-activating (methylalkyl)succinate synthases to the discovery of bacterial groups previously unknown to possess alkyl-/arylalkylsuccinate synthases by means of functional gene markers and in situ field studies enabled by state-of-the-art stable isotope probing and fractionation approaches. Other topics are Mo-cofactor-dependent dehydrogenases performing O₂-independent hydroxylation of hydrocarbons and alkyl side chains (ethylbenzene, p-cymene, cholesterol, n-hexadecane), degradation of p-alkylated benzoates and toluenes, glycyl radical-bearing 4-hydroxyphenylacetate decarboxylase, novel types of carboxylation reactions (for acetophenone, acetone, and potentially also benzene and naphthalene), W-cofactor-containing enzymes for reductive dearomatization of benzoyl-CoA (class II benzoyl-CoA reductase) in obligate anaerobes and addition of water to acetylene, fermentative formation of cyclohexanecarboxylate from benzoate, and methanogenic degradation of hydrocarbons.

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Substrate-bound Structures of Benzylsuccinate Synthase Reveal How Toluene Is Activated in Anaerobic Hydrocarbon Degradation

Cited by 40 publications

References 42 publications

Molecular Basis of C–N Bond Cleavage by the Glycyl Radical Enzyme Choline Trimethylamine-Lyase

Molecular Basis of C–N Bond Cleavage by the Glycyl Radical Enzyme Choline Trimethylamine-Lyase

New tricks for the glycyl radical enzyme family

Anaerobic Microbial Degradation of Hydrocarbons: From Enzymatic Reactions to the Environment

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