A Continuing Career in Biocatalysis: Frances H. Arnold

On the occasion of Professor Frances H. Arnold’s recent acceptance of the 2018 Nobel Prize in Chemistry, we honor her numerous contributions to the fields of directed evolution and biocatalysis. Arnold pioneered the development of directed evolution methods for engineering enzymes as biocatalysts. Her highly interdisciplinary research has provided grounds not only for understanding the mechanisms of enzyme evolution but also for developing commercially viable enzyme biocatalysts and biocatalytic processes. In this Account, we highlight some of her notable contributions in the past three decades in the development of foundational directed evolution methods and their applications in the design and engineering of enzymes with desired functions for biocatalysis. Her work has created a paradigm shift in the broad catalysis field

Directed evolution of cytochrome c for carbon–silicon bond formation: Bringing silicon to life

Organic compounds containing silicon are important for a number of applications, from polymers to semiconductors. The catalysts used for creating carbon-silicon bonds, however, often require expensive trace metals or have limited lifetimes. Borrowing from the ability of some metallo-enzymes to catalyze other rare carbene insertion reactions, Kan et al. used heme proteins to form carbon-silicon bonds across a range of conditions and substrates (see the Perspective by Klare and Oestreich). Directed evolution experiments using cytochrome c from Rhodothermus marinus improved the reaction to be 15 times more efficient than industrial catalysts

A Continuing Career in Biocatalysis: Frances H. Arnold

On the occasion of Professor Frances H. Arnold’s recent acceptance of the 2018 Nobel Prize in Chemistry, we honor her numerous contributions to the fields of directed evolution and biocatalysis. Arnold pioneered the development of directed evolution methods for engineering enzymes as biocatalysts. Her highly interdisciplinary research has provided grounds not only for understanding the mechanisms of enzyme evolution but also for developing commercially viable enzyme biocatalysts and biocatalytic processes. In this Account, we highlight some of her notable contributions in the past three decades in the development of foundational directed evolution methods and their applications in the design and engineering of enzymes with desired functions for biocatalysis. Her work has created a paradigm shift in the broad catalysis field

Machine learning-assisted directed protein evolution with combinatorial libraries

To reduce experimental effort associated with directed protein evolution and to explore the sequence space encoded by mutating multiple positions simultaneously, we incorporate machine learning in the directed evolution workflow. Combinatorial sequence space can be quite expensive to sample experimentally, but machine learning models trained on tested variants provide a fast method for testing sequence space computationally. We validate this approach on a large published empirical fitness landscape for human GB1 binding protein, demonstrating that machine learning-guided directed evolution finds variants with higher fitness than those found by other directed evolution approaches. We then provide an example application in evolving an enzyme to produce each of the two possible product enantiomers (stereodivergence) of a new-to-nature carbene Si-H insertion reaction. The approach predicted libraries enriched in functional enzymes and fixed seven mutations in two rounds of evolution to identify variants for selective catalysis with 93% and 79% ee. By greatly increasing throughput with in silico modeling, machine learning enhances the quality and diversity of sequence solutions for a protein engineering problem.Comment: Corrected best S-selective variant sequence in Figure 4. Corrected less R-selective variant sequences from Round II Input library in Table 2 and Supp Table 4. Corrections may also be found on PNAS version https://www.pnas.org/content/early/2019/12/26/192177011

A Biocatalytic Platform for Synthesis of Chiral α-Trifluoromethylated Organoborons

There are few biocatalytic transformations that produce fluorine-containing molecules prevalent in modern pharmaceuticals. To expand the scope of biocatalysis for organofluorine synthesis, we have developed an enzymatic platform for highly enantioselective carbene B–H bond insertion to yield versatile α-trifluoromethylated (α-CF_3) organoborons, an important class of organofluorine molecules that contain stereogenic centers bearing both CF_3 and boron groups. In contrast to current “carbene transferase” enzymes that use a limited set of simple diazo compounds as carbene precursors, this system based on Rhodothermus marinus cytochrome c (Rma cyt c) can accept a broad range of trifluorodiazo alkanes and deliver versatile chiral α-CF_3 organoborons with total turnovers up to 2870 and enantiomeric ratios up to 98.5:1.5. Computational modeling reveals that this broad diazo scope is enabled by an active-site environment that directs the alkyl substituent on the heme CF_3-carbene intermediate toward the solvent-exposed face, thereby allowing the protein to accommodate diazo compounds with diverse structural features

Diversity-Oriented Enzymatic Synthesis of Cyclopropane Building Blocks

While biocatalysis is increasingly incorporated into drug development pipelines, it is less commonly used in the early stages of drug discovery. By engineering a protein to produce a chiral motif with a derivatizable functional handle, biocatalysts can be used to help generate diverse building blocks for drug discovery. Here we show the engineering of two variants of Rhodothermus marinus nitric oxide dioxygenase (RmaNOD) to catalyze the formation of cis- and trans-diastereomers of a pinacolboronate-substituted cyclopropane which can be readily derivatized to generate diverse stereopure cyclopropane building blocks

Diversity-Oriented Enzymatic Synthesis of Cyclopropane Building Blocks

While biocatalysis is increasingly incorporated into drug development pipelines, it is less commonly used in the early stages of drug discovery. By engineering a protein to produce a chiral motif with a derivatizable functional handle, biocatalysts can be used to help generate diverse building blocks for drug discovery. Here we show the engineering of two variants of Rhodothermus marinus nitric oxide dioxygenase (RmaNOD) to catalyze the formation of cis- and trans-diastereomers of a pinacolboronate-substituted cyclopropane which can be readily derivatized to generate diverse stereopure cyclopropane building blocks

Directed evolution of cytochrome c for carbon–silicon bond formation: Bringing silicon to life

Organic compounds containing silicon are important for a number of applications, from polymers to semiconductors. The catalysts used for creating carbon-silicon bonds, however, often require expensive trace metals or have limited lifetimes. Borrowing from the ability of some metallo-enzymes to catalyze other rare carbene insertion reactions, Kan et al. used heme proteins to form carbon-silicon bonds across a range of conditions and substrates (see the Perspective by Klare and Oestreich). Directed evolution experiments using cytochrome c from Rhodothermus marinus improved the reaction to be 15 times more efficient than industrial catalysts

Engineered Cytochrome c-Catalyzed Lactone-Carbene B–H Insertion

Previous work has demonstrated that variants of a heme protein, Rhodothermus marinus cytochrome c (Rma cyt c), catalyze abiological carbene boron–hydrogen (B–H) bond insertion with high efficiency and selectivity. Here we investigated this carbon–boron bond-forming chemistry with cyclic, lactone-based carbenes. Using directed evolution, we obtained a Rma cyt c variant BOR^(LAC) that shows high selectivity and efficiency for B–H insertion of 5- and 6-membered lactone carbenes (up to 24,500 total turnovers and 97.1:2.9 enantiomeric ratio). The enzyme shows low activity with a 7-membered lactone carbene. Computational studies revealed a highly twisted geometry of the 7-membered lactone carbene intermediate relative to 5- and 6-membered ones. Directed evolution of cytochrome c together with computational characterization of key iron-carbene intermediates has allowed us to expand the scope of enzymatic carbene B–H insertion to produce new lactone-based organoborons