A Continuing Career in Biocatalysis: Frances H. Arnold

Abstract

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 a ground 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.

Keywords: C–H functionalization; P450s; abiological functions; biocatalysis; carbene transfer reactions; directed evolution; enzyme engineering; nitrene transfer reaction.

Figure 1.

Workflow of a typical directed evolution experiment.

Figure 2.. Directed evolution of a cytochrome P450 propane monooxygenase.

The long-chain fatty acid hydroxylase P450 BM3 from Bacillus megaterium was converted into a highly efficient propane monooxygenase (P450_PMO) over 13 rounds of directed evolution.^{, ,} The evolved enzyme accumulated 3 beneficial mutations in the reductase domain and 20 in the heme domain (yellow, sphere models), many of which are distant from the heme cofactor (red, sphere model). Refinement of catalytic efficiency for propane oxidation, an activity not exhibited by wild-type P450 BM3, also involved a profound reshaping and partitioning of the substrate access pathway (inserts).

Figure 3.

Catalytic and stability properties (a) and substrate profile (b) of selected variants along the P450_PMO lineage. Total turnover numbers (TTN), coupling efficiency (mol oxidized product/mol oxidized NADPH), and kinetic properties (K_M, k_cat) refer to propane oxidation. Thermal stability is shown as T₅₀ (half-maximal inactivation temperature after 10-min incubation). Substrate profile is reported as relative activity on C1–C10 alkane series. Adapted with permission from ref. 41. Copyright 2008, Elsevier.

Figure 4.. Directed evolution of enzymes using structure-based recombination.

(a) Generation of chimeric enzymes via SCHEMA-guided recombination of fragments derived from homologous parent sequences and chosen to minimize structural disruption. (b) SCHEMA-guided recombination of β-lactamase PSE-2 and TEM-1 produces a higher fraction of functional chimeras compared to random mutagenesis. (c) Chimeric P450s obtained via SCHEMA-guided recombination of P450 BM3 (CYP102A1) with its homologous enzymes CYP102A2 and CYP102A3 can exhibit higher stability against thermal denaturation compared to any of the parent P450s. Adapted with permission from ref. 62. Copyright 2005, National Academy of Sciences.

Scheme 1.

Laboratory-evolved P450 BM3 variants for synthetic applications: (a) selective epoxidation of terminal alkenes; (b) stereoselective synthesis of mandelic acid derivatives; (c) generation of human drug metabolites (sites hydroxylated by the engineered P450 BM3 variants are indicated by arrows); and (d) regioselective demethylation of permethylated monosaccharides. TON = turnover number; ee = enantiomeric excess; select. = selectivity.

Scheme 2.

Engineered heme protein-catalyzed new-to-nature transformations: (a) synthesis of the cyclopropane core of antidepressant levomilnacipran; (b) enzymatic stereodivergent cyclopropanation of octene catalyzed by a suite of engineered cytochrome P411 proteins and globins.

Scheme 3.

Cytochrome P411_BM3-catalyzed bicyclobutanation and cyclopropenation.

Scheme 4.

Engineered R. marinus cytochrome c-catalyzed carbon–silicon and carbon–boron bond formation.^,

Scheme 5.

C–H functionalization enabled by enzymatic nitrene and carbene transfer C–H insertion: (a) regioselective cyclization via intramolecular nitrene insertion into benzylic C–H bonds; (b) divergent synthesis of β-, γ-, and δ-lactams by P411_BM3-catalyzed intramolecular C–H amidation; (c) P411_BM3-catalyzed intermolecular C–H amination; (d) P411_BM3-catalyzed intermolecular C–H alkylation.