Mechanistic Studies of a Skatole-Forming Glycyl Radical Enzyme Suggest Reaction Initiation via Hydrogen Atom Transfer.

Gut microbial decarboxylation of amino acid-derived arylacetates is a chemically challenging enzymatic transformation which generates small molecules that impact host physiology. The glycyl radical enzyme (GRE) indoleacetate decarboxylase from Olsenella uli (Ou IAD) performs the non-oxidative radical decarboxylation of indole-3-acetate (I3A) to yield skatole, a disease-associated metabolite produced in the guts of swine and ruminants. Despite the importance of IAD, our understanding of its mechanism is limited. Here, we characterize the mechanism of Ou IAD, evaluating previously proposed hypotheses of: (1) a Kolbe-type decarboxylation reaction involving an initial 1-e- oxidation of the carboxylate of I3A or (2) a hydrogen atom abstraction from the α-carbon of I3A to generate an initial carbon-centered radical. Site-directed mutagenesis, kinetic isotope effect experiments, analysis of reactions performed in D2O, and computational modeling are consistent with a mechanism involving initial hydrogen atom transfer. This finding expands the types of radical mechanisms employed by GRE decarboxylases and non-oxidative decarboxylases, more broadly. Elucidating the mechanism of IAD decarboxylation enhances our understanding of radical enzymes and may inform downstream efforts to modulate this disease-associated metabolism.

Biocatalytic Transformations of Silicon-the Other Group 14 Element.

Significant inroads have been made using biocatalysts to perform new-to-nature reactions with high selectivity and efficiency. Meanwhile, advances in organosilicon chemistry have led to rich sets of reactions holding great synthetic value. Merging biocatalysis and silicon chemistry could yield new methods for the preparation of valuable organosilicon molecules as well as the degradation and valorization of undesired ones. Despite silicon's importance in the biosphere for its role in plant and diatom construction, it is not known to be incorporated into any primary or secondary metabolites. Enzymes have been found that act on silicon-containing molecules, but only a few are known to act directly on silicon centers. Protein engineering and evolution has and could continue to enable enzymes to catalyze useful organosilicon transformations, complementing and expanding upon current synthetic methods. The role of silicon in biology and the enzymes that act on silicon-containing molecules are reviewed to set the stage for a discussion of where biocatalysis and organosilicon chemistry may intersect.

Discovering radical-dependent enzymes in the human gut microbiota.

Human gut microbes have a tremendous impact on human health, in part due to their unique chemical capabilities. In the anoxic environment of the healthy human gut, many important microbial metabolic transformations are performed by radical-dependent enzymes. Although identifying and characterizing these enzymes has been challenging, recent advances in genome and metagenome sequencing have enabled studies of their chemistry and biology. Focusing on the glycyl radical enzyme family, one of the most enriched protein families in the human gut microbiota, we highlight different approaches for discovering radical-dependent enzymes that influence host health and disease.

Characterization of 1,2-Propanediol Dehydratases Reveals Distinct Mechanisms for B12-Dependent and Glycyl Radical Enzymes.

Propanediol dehydratase (PD), a recently characterized member of the glycyl radical enzyme (GRE) family, uses protein-based radicals to catalyze the chemically challenging dehydration of ( S)-1,2-propanediol. This transformation is also performed by the well-studied enzyme B12-dependent propanediol dehydratase (B12-PD) using an adenosylcobalamin cofactor. Despite the prominence of PD in anaerobic microorganisms, it remains unclear if the mechanism of this enzyme is similar to that of B12-PD. Here we report 18O labeling experiments that suggest PD and B12-PD employ distinct mechanisms. Unlike B12-PD, PD appears to catalyze the direct elimination of a hydroxyl group from an initially formed substrate-based radical, avoiding the generation of a 1,1- gem diol intermediate. Our studies provide further insights into how GREs perform elimination chemistry and highlight how nature has evolved diverse strategies for catalyzing challenging reactions.

Perfluoro-tert-butyl-homoserine as a sensitive 19F NMR reporter for peptide-membrane interactions in solution.

Fluorine ((19)F) NMR is a valuable tool for studying dynamic biological processes. However, increasing the sensitivity of fluorinated reporter molecules is a key to reducing acquisition times and accessing transient biological interactions. Here, we evaluate the utility a novel amino acid, L-O-(perfluoro-t-butyl)-homoserine (pFtBSer), that can easily be synthesized and incorporated into peptides and provides greatly enhanced sensitivity over currently used (19)F biomolecular NMR probes. Incorporation of pFtBSer into the potent antimicrobial peptide MSI-78 results in a sharp (19)F NMR singlet that can be readily detected at concentrations of 5 µm and lower. We demonstrate that pFtBSer incorporation into MSI-78 provides a sensitive tool to study binding through (19)F NMR chemical shift and nuclear relaxation changes. These results establish future potential for pFtBSer to be incorporated into various proteins where NMR signal sensitivity is paramount, such as in-cell investigations.

Influence of fluorination on the thermodynamics of protein folding.

The introduction of highly fluorinated analogues of hydrophobic amino acid residues into proteins has proved an effective and general strategy for increasing protein stability toward both chemical denaturants and heat. However, the thermodynamic basis for this stabilizing effect, whether enthalpic or entropic in nature, has not been extensively investigated. Here we describe studies in which the values of ΔH°, ΔS°, and ΔCp° have been determined for the unfolding of a series of 12 small, de novo-designed proteins in which the hydrophobic core is packed with various combinations of fluorinated and non-fluorinated amino acid residues. The increase in the free energy of unfolding with increasing fluorine content is associated with increasingly unfavorable entropies of unfolding and correlates well with calculated changes in apolar solvent-accessible surface area. ΔCp° for unfolding is positive for all the proteins and, similarly, correlates with changes in apolar solvent-accessible surface area. ΔH° for unfolding shows no correlation with either fluorine content or changes in apolar solvent-accessible surface area. We conclude that conventional hydrophobic effects adequately explain the enhanced stabilities of most highly fluorinated proteins. The extremely high thermal stability of these proteins results, in part, from their very low per-residue ΔCp°, as has been observed for natural thermostable proteins.