Research

We are also developing new approaches to construct and screen libraries, including using techniques such as continuous evolution and fluorescence activated cell sorting. While these approaches are more limited in the range of compounds and reactions to which they can be applied, they have the potential to dramatically accelerate the evolution process and thus allow us to probe different questions regarding enzyme evolution and catalysis.

As noted above, directed evolution of proteins involves iterative mutation of a protein and functional screening/selection of the resulting variants to create protein variants with improved properties. Prior to the development of directed evolution, protein engineering, such as it was, largely involved targeted point mutation of proteins and thus only modest improvements in function. Directed evolution revolutionized this process and has led to stunning improvements in protein function for fine/commodity chemical synthesis, selective catalysis, pharmaceuticals development, materials science, chemical biology, and a wide range of other fields. It is safe to say that any application that uses a protein could benefit from some form of directed evolution at some point in its development. Moreover, directed evolution has influenced the means by which protein function and natural evolution is understood and studied. Two seminal and complementary methods, one by Prof. Frances Arnold published in 1993 and one by Dr. Willemm (Pim) Stemmer (deceased) published in 1994, mark the advent of directed evolution, and the pair received the 2011 Draper Prize for their discoveries. One half of the 2018 Nobel Prize in Chemistry was awarded to Prof. Arnold for her ground-breaking work on “the directed evolution of enzymes”. Prof. Lewis was a postdoctoral fellow with Prof. Arnold at Caltech from 2008-2010.

Biocatalysis

We are exploiting this property to engineer various flavin dependent halogenases (FDHs) for use in organic synthesis due to the importance of halogenated compounds as both building blocks and active pharmaceutical ingredients. Our group has reported the first examples in which directed evolution of FDHs (typically the tryptophan halogenase RebH) has been used to improve stability, expand substrate scope (Fig. 1A), achieve high selectivity for different sites on a given substrate (Fig. 1B), and enable enantioselective (Fig. 1C)and atroposelective (in progress) transformations. Recently, we also found that evolved FDHs can catalyze non-native enantioselective halocyclization (Fig. 1D), providing access to halogenated aliphatic compounds rather than the aromatic compounds generated via native FDH catalysis. We have used genome mining (Fig. 1E) to identify FDHs that provide unique function relative to our evolved enzymes, further expanding the utility of FDHs for chemical synthesis. Finally, we explore structure function relationships in improved enzymes to help rationalize mechanisms for the observed improvements and to inform subsequent engineering efforts.  More recently, we have been exploring polymer degradation by lipases and non-native enzyme catalysis by B12-dependent proteins and enzymes and Fe(II)- and α-ketoglutarate-dependent oxygenases and halogenases (in progress). 

Artificial Metalloenzymes

Many powerful reactions, particularly those catalyzed by non-biological metals, are not found in nature, so systems that combine the reactivity of metal catalysts with the evolvability and specificity of enzymes are highly sought after.To expand the scope of reactions are developing new classes of artificial metalloenzymes (ArMs), hybrid constructs comprised of protein scaffolds and metal catalysts. Our group pioneered the use of strain-promoted azide alkyne addition using genetically-encoded azide residues to facilitate ArM formation (Fig. 2A) using a variety of cofactors (Fig. 2B). We have subsequently focused on ArMs generated by linking these cofactors to a prolyl oligopeptidase (POP) from Pyrococcus furious. For example, optimization of dirhodium ArMs generated from cofactor 1 using both rational design (Fig. 2C) and directed evolution (Fig. 2D/E) is being used to produce highly active enzymes for a variety of carbene addition and insertion reactions (Fig. 2F). We recently showed that directed evolution can also be used to engineer dirhodium ArMs to enable a cascade process with an ene reductase that otherwise requires intermediate isolation. Very recently we showed that we could incorporate ruthenium cofactor 2 into POP to modulate its photophysical properties, and, in a related effort, use genetically encoded bipyridine residues to control the conformation and native peptidase catalysis of POP.

In parallel with our ArM design and evolution efforts, we are pursuing detailed mechanistic studies on the systems developed in our laboratory to date. These studies involve steady state kinetics, biophysical experiments (e.g. FRET, LC-MS/MS, NMR, UV/Vis, etc.), and computational simulation (MD, QM/MM in collaboration with the Roux group at Chicago). Beyond shedding light on the function of these complex and fascinating systems, these studies are intended to inform future design efforts aimed at improving ArM activity and selectivity for synthetic applications.

Transition Metal Catalysis

Historically, bioinorganic chemists have sought to synthesize coordination complexes that mimic the structures, spectroscopic features, and even functions of metalloenzyme cofactors that must otherwise be studied, often with great difficulty, in the context of their host enzymes. A key lesson from these efforts is that enzymes play enormous roles in modulating the structures and functions of their metallocofactors. This lesson suggests that ArMs generated by incorporating well-characterized, stable catalysts into protein scaffolds (as outlined above) represent only a subset of the structures that could be possible. For example, in a reversal of the classical bioinorganic chemistry paradigm, proteins could be used to stabilize structures that cannot be readily accessed in solution or maintained throughout a catalytic cycle. Mechanistic studies on small molecule transition metal catalysts could inform the design of protein scaffolds that provide access to putative intermediates to both verify their catalytic relevance and improve their catalytic properties. Our first forays into this area are focused on mono- and dipalladium complexes inspired by our finding that many reactions catalyzed by mixtures of Pd(II) salts and mono-protected amino acid (MPAA) ligands are actually catalyzed by MPAA-bridged Pd(II) dimers. These efforts are in their infancy but progressing rapidly, so please check back to see how this project evolves.