Engineering the Reaction Pathway of a Non-heme Iron Oxygenase Using Ancestral Sequence Reconstruction.

Non-heme iron (Fe), α-ketoglutarate (α-KG)-dependent oxygenases are a family of enzymes that catalyze an array of transformations that cascade forward after the formation of radical intermediates. Achieving control over the reaction pathway is highly valuable and a necessary step toward broadening the applications of these biocatalysts. Numerous approaches have been used to engineer the reaction pathway of Fe/α-KG-dependent enzymes, including site-directed mutagenesis, DNA shuffling, and site-saturation mutagenesis, among others.

Ancestral Sequence Reconstruction to Enable Biocatalytic Synthesis of Azaphilones.

Biocatalysis can be powerful in organic synthesis but is often limited by enzymes' substrate scope and selectivity. Developing a biocatalytic step involves identifying an initial enzyme for the target reaction followed by optimization through rational design, directed evolution, or both. These steps are time consuming, resource-intensive, and require expertise beyond typical organic chemistry.

Biocatalytic Stereoselective Oxidation of 2-Arylindoles.

3-Hydroxyindolenines can be used to access several structural motifs that are featured in natural products and pharmaceutical compounds, yet the chemical synthesis of 3-hydroxyindolenines is complicated by overoxidation, rearrangements, and complex product mixtures. The selectivity possible in enzymatic reactions can overcome these challenges and deliver enantioenriched products. Herein, we present the development of an asymmetric biocatalytic oxidation of 2-arylindole substrates aided by a curated library of flavin-dependent monooxygenases (FDMOs) sampled from an ancestral sequence space, a sequence similarity network, and a deep-learning-based latent space model.

Stereodynamic Strategies to Induce and Enrich Chirality of Atropisomers at a Late Stage.

Enantiomers, where chirality arises from restricted rotation around a single bond, are atropisomers. Due to the unique nature of the origins of their chirality, synthetic strategies to access these compounds in an enantioselective manner differ from those used to prepare enantioenriched compounds containing point chirality arising from an unsymmetrically substituted carbon center. In particular stereodynamic transformations, such as dynamic kinetic resolutions, thermodynamic dynamic resolutions, and deracemizations, which rely on the ability to racemize or interconvert enantiomers, are a promising set of transformations to prepare optically pure compounds in the late stage of a synthetic sequence.

Deciphering the evolution of flavin-dependent monooxygenase stereoselectivity using ancestral sequence reconstruction.

Controlling the selectivity of a reaction is critical for target-oriented synthesis. Accessing complementary selectivity profiles enables divergent synthetic strategies, but is challenging to achieve in biocatalytic reactions given enzymes' innate preferences of a single selectivity. Thus, it is critical to understand the structural features that control selectivity in biocatalytic reactions to achieve tunable selectivity.

Effect of lysine methylation and acetylation on the RNA recognition and cellular uptake of Tat-derived peptides.

The two lysine (Lys) residues in the human immunodeficiency virus trans-activator of transcription protein (HIV Tat protein) basic region (residues 47-57) are crucial for two bioactivities: RNA recognition and cellular uptake. Since the post-translational modifications of these two Lys residues affect the biological function of the Tat protein, we investigated the effect of methylation and acetylation of Lys50 and Lys51 in Tat-derived peptides on the two bioactivities. Tat-derived peptides, in which each lysine was replaced with a methylated- or acetylated-Lys, were synthesized by solid phase peptide synthesis.