Intramolecular Cyclization and a Retro-Ene Reaction Enable the Rapid Fragmentation of a Vitamin B1-derived Breslow Intermediate.

In solution, analogues of the Breslow intermediate formed during catalysis by benzoylformate decarboxylase (BFDC) undergo rapid, irreversible fragmentation. The ability of BFDC to prevent this reaction and preserve its' cofactor is a striking example of an enzyme 'steering' a reactive intermediate towards a productive pathway. To understand how BFDC suppresses the off-pathway reactivity of this Breslow intermediate, a clear mechanistic understanding of the fragmentation reaction is required. Here, DFT calculations reveal an unexpected mechanism for the solution-phase fragmentation that involves an intramolecular cyclization and a subsequent retro-ene reaction to release the final products. Free energy profiles demonstrate that this pathway is significantly more facile than the previously proposed mechanism that invoked Breslow intermediate enolates as intermediates. Additional computations have been performed to understand why related Breslow intermediates do not undergo analogous fragmentation reactions. Calculations performed with two closely related Breslow intermediates suggest that subtle differences in the relative values of ∆G‡ for protonation and fragmentation dictate whether a given intermediate will fragment or not. These differences and the fragmentation mechanism unveiled in this work may have ramifications for the mechanism of BFDC and other thiamin-dependent enzymes and could provide general lessons related to the control of reactive intermediates by enzymes.

Discovery of a Tambjamine Gene Cluster in Streptomyces Suggests Convergent Evolution in Bipyrrole Natural Product Biosynthesis.

While bacterial natural products are a valuable source of therapeutics, the molecules produced by most biosynthetic gene clusters remain unknown. Tambjamine YP1, produced by Pseudoalteromonas tunicata, is partially derived from fatty acids siphoned from primary metabolism. A structurally similar tambjamine produced by Streptomyces, BE-18591, had not been linked to a gene cluster. Using enzymes putatively implicated in the construction of these two tambjamines, we used sequence similarity networks and gene knockout experiments to identify the biosynthetic gene cluster responsible for the production of tambjamine BE-18591 in Streptomyces albus. Despite the structural similarities between YP1 and BE-18591, the biosynthesis of the alkylamine tails of these molecules differs significantly, with the S. albus gene cluster putatively encoding a dedicated system for the construction of the fatty acid precursor to BE-18591. These different pathways in Pseudoalteromonas and Streptomyces suggest that evolutionary convergence is operative, with similar selective pressures leading to the emergence of structurally similar tambjamine natural products using different biosynthetic logic.

Sulfite-Catalyzed Nucleophilic Substitution Reactions with Thiamin and Analogous Pyrimidine Donors Proceed via an SNAE Mechanism.

When treated with SO32-, thiamin undergoes a substitution reaction to release a thiazole leaving group and the corresponding sulfonate. Although this reaction could proceed via a simple SN2-like mechanism, a multistep addition-elimination (SNAE) mechanism involving the addition of SO32- to C6' of the 4-aminopyrimidine of thiamin has also been proposed. Although this reaction has potential utility in the synthesis of substituted pyrimidines and provides a direct analogue to reactions catalyzed by thiaminases, a detailed mechanistic picture of the SO32--catalyzed cleavage of thiamin has remained elusive. Here, DFT calculations have been used to probe the relative energetics and the factors that shape the potential energy surfaces that define the possible mechanisms of substitution. These calculations provide clear support for the SNAE mechanism over an SN2-like process and illustrate that the unique ability of SO32- to activate thiamin toward nucleophilic displacement is due to the combined nucleophilicity and relatively poor leaving group ability of SO32-. Both of these factors favor the forward partitioning of the sulfite adduct toward the cleavage products whereas adducts formed with other nucleophiles overwhelmingly revert to reactants. Calculations performed with a range of substrates with various electrophilicities and nucleofugalities consistently suggest that the SNAE pathway is significantly lower in energy than the direct substitution, illustrating that this SO32--catalyzed multistep process is likely to be broadly applicable both in solution and in catalysis by thiaminases.

The convergence of bacterial natural products from evolutionarily distinct pathways.

As bacteria readily convert simple starting materials into a diverse array of complex molecules with useful bioactivities, these microorganisms and their biosynthetic machinery represent attractive alternatives to traditional chemical syntheses. While the well-documented divergent evolution of biosynthesis has allowed bacteria to explore wide swaths of natural product chemical space, the convergent evolution of these pathways remains a comparably rare phenomenon. The emergence of similar phenotypes within disparate genetic contexts provides a unique opportunity to probe the limitations of natural selection and the predictability and reproducibility of evolution under different constraints. Here, we report several recent examples of functional and structural convergence of bacterial natural products, as well as intra- and inter-domain convergence of bacterial biosynthetic machinery. While the genetic underpinnings of biosynthetic pathway evolution are of fundamental interest, the evolutionary constraints exemplified by phenotypic convergence also have immediate implications for efforts to engineer microorganisms for therapeutic small molecule production.