Sarah A. Shepherd

Development of Halogenase Enzymes for Use in Synthesis

Nature has evolved halogenase enzymes to regioselectively halogenate a diverse range of biosynthetic precursors, with the halogens introduced often having a profound effect on the biological activity of the resulting natural products. Synthetic endeavors to create non-natural bioactive small molecules for pharmaceutical and agrochemical applications have also arrived at a similar conclusion: halogens can dramatically improve the properties of organic molecules for selective modulation of biological targets in vivo. Consequently, a high proportion of pharmaceuticals and agrochemicals on the market today possess halogens. Halogenated organic compounds are also common intermediates in synthesis and are particularly valuable in metal-catalyzed cross-coupling reactions. Despite the potential utility of organohalogens, traditional nonenzymatic halogenation chemistry utilizes deleterious reagents and often lacks regiocontrol. Reliable, facile, and cleaner methods for the regioselective halogenation of organic compounds are therefore essential in the development of economical and environmentally friendly industrial processes. A potential avenue toward such methods is the use of halogenase enzymes, responsible for the biosynthesis of halogenated natural products, as biocatalysts. This Review will discuss advances in developing halogenases for biocatalysis, potential untapped sources of such biocatalysts and how further optimization of these enzymes is required to achieve the goal of industrial scale biohalogenation.

A structure-guided switch in the regioselectivity of a tryptophan halogenase

Flavin‐dependent halogenases are potentially useful biocatalysts for the regioselective halogenation of aromatic compounds. Haloaromatic compounds can be utilised in the synthesis and biosynthesis of pharmaceuticals and other valuable products. Here we report the first X‐ray crystal structure of a tryptophan 6‐halogenase (SttH), which enabled key residues that contribute to the regioselectivity in tryptophan halogenases to be identified. Structure‐guided mutagenesis resulted in a triple mutant (L460F/P461E/P462T) that exhibited a complete switch in regioselectivity; with the substrate 3‐indolepropionate 75 % 5‐chlorination was observed with the mutant in comparison to 90 % 6‐chlorination for the wild‐type SttH. This is the first clear example of how regiocomplementary halogenases can be created from a single parent enzyme. The biocatalytic repertoire of SttH was also expanded to include a range of indolic and non‐indolic substrates.

An Enzyme Cascade for Selective Modification of Tyrosine Residues in Structurally Diverse Peptides and Proteins

Bioorthogonal chemistry enables a specific moiety in a complex biomolecule to be selectively modified in the presence of many reactive functional groups and other cellular entities. Such selectivity has become indispensable in biology, enabling biomolecules to be derivatized, conjugated, labeled, or immobilized for imaging, biochemical assays, or therapeutic applications. Methyltransferase enzymes (MTase) that accept analogues of the cofactor S-adenosyl methionine have been widely deployed for alkyl-diversification and bioorthogonal labeling. However, MTases typically possess tight substrate specificity. Here we introduce a more flexible methodology for selective derivatization of phenolic moieties in complex biomolecules. Our approach relies on the tandem enzymatic reaction of a fungal tyrosinase and the mammalian catechol-O-methyltransferase (COMT), which can effect the sequential hydroxylation of the phenolic group to give an intermediate catechol moiety that is subsequently O-alkylated. When used in this combination, the alkoxylation is highly selective for tyrosine residues in peptides and proteins, yet remarkably tolerant to changes in the peptide sequence. Tyrosinase-COMT are shown to provide highly versatile and regioselective modification of a diverse range of substrates including peptide antitumor agents, hormones, cyclic peptide antibiotics, and model proteins.

RadH : a versatile halogenase for integration into synthetic pathways

Abstract Flavin‐dependent halogenases are useful enzymes for providing halogenated molecules with improved biological activity, or intermediates for synthetic derivatization. We demonstrate how the fungal halogenase RadH can be used to regioselectively halogenate a range of bioactive aromatic scaffolds. Site‐directed mutagenesis of RadH was used to identify catalytic residues and provide insight into the mechanism of fungal halogenases. A high‐throughput fluorescence screen was also developed, which enabled a RadH mutant to be evolved with improved properties. Finally we demonstrate how biosynthetic genes from fungi, bacteria, and plants can be combined to encode a new pathway to generate a novel chlorinated coumarin “non‐natural” product in E. coli.

A High‐Throughput Assay for Arylamine Halogenation Based on a Peroxidase‐Mediated Quinone–Amine Coupling with Applications in the Screening of Enzymatic Halogenations

Arylhalides are important building blocks in many fine chemicals, pharmaceuticals and agrochemicals, and there has been increasing interest in the development of more “green” halogenation methods based on enzyme catalysis. However, the screening and development of new enzymes for biohalogenation has been hampered by a lack of high-throughput screening methods. Described herein is the development of a colorimetric assay for detecting both chemical and enzymatic arylamine halogenation reactions in an aqueous environment. The assay is based on the unique UV/Vis spectrum created by the formation of an ortho-benzoquinone-amine adduct, which is produced by the peroxidase-catalysed benzoquinone generation, followed by Michael addition of either a halogenated or non-halogenated arylamine. This assay is sensitive, rapid and amenable to high-throughput screening platforms. We have also shown this assay to be easily coupled to a flavin-dependent halogenase, which currently lacks any convenient colorimetric assay, in a “one-pot” workflow.

Structure and biocatalytic scope of thermophilic flavin-dependent halogenase and flavin reductase enzymes

Flavin-dependent halogenase (Fl-Hal) enzymes have been shown to halogenate a range of synthetic as well as natural aromatic compounds. The exquisite regioselectively of Fl-Hal enzymes can provide halogenated building blocks which are inaccessible using standard halogenation chemistries. Consequently, Fl-Hal are potentially useful biocatalysts for the chemoenzymatic synthesis of pharmaceuticals and other valuable products, which are derived from haloaromatic precursors. However, the application of Fl-Hal enzymes, in vitro, has been hampered by their poor catalytic activity and lack of stability. To overcome these issues, we identified a thermophilic tryptophan halogenase (Th-Hal), which has significantly improved catalytic activity and stability, compared with other Fl-Hal characterised to date. When used in combination with a thermostable flavin reductase, Th-Hal can efficiently halogenate a number of aromatic substrates. X-ray crystal structures of Th-Hal, and the reductase partner (Th-Fre), provide insights into the factors that contribute to enzyme stability, which could guide the discovery and engineering of more robust and productive halogenase biocatalysts.

Effects of Active-Site Modification and Quaternary Structure on the Regioselectivity of Catechol-O-Methyltransferase.

Abstract Catechol‐O‐methyltransferase (COMT), an important therapeutic target in the treatment of Parkinsons disease, is also being developed for biocatalytic processes, including vanillin production, although lack of regioselectivity has precluded its more widespread application. By using structural and mechanistic information, regiocomplementary COMT variants were engineered that deliver either meta‐ or para‐methylated catechols. X‐ray crystallography further revealed how the active‐site residues and quaternary structure govern regioselectivity. Finally, analogues of AdoMet are accepted by the regiocomplementary COMT mutants and can be used to prepare alkylated catechols, including ethyl vanillin.

Recent Advances in Methyltransferase Biocatalysis

S-adenosyl-L-methionine-dependent methyltransferses are ubiquitous in nature, methylating a vast range of small molecule metabolites, as well as biopolymers. This review covers the recent advances in the development of methyltransferase enzymes for synthetic applications, focusing on the methyltransferase catalyzed transformations with S-adenosyl methionine analogs, as well as non-native substrates. We discuss how metabolic engineering approaches have been used to enhance S-adenosyl methionine production in vivo. Enzymatic approaches that enable the more efficient generation of S-adenosyl methionine analogs, including more stable analogs, will also be described; this has expanded the biocatalytic repertoire of methyltransferases from methylation to a broader range of alkylation reactions. The review also examines how the selectivity of the methyltransferase enzymes can be improved through structure guided mutagenesis approaches. Finally, we will discuss how methyltransferases can be deployed in multi-enzyme cascade reactions and suggest future challenges and avenues for further investigation.