Review Halogenase
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catalysts Review Recent Advances in Flavin-Dependent Halogenase Biocatalysis: Sourcing, Engineering, and Application Johannes Büchler y, Athena Papadopoulou y and Rebecca Buller * Department of Life Sciences and Facility Management, Competence Center for Biocatalysis, Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences, Einsiedlerstrasse 31, 8820 Waedenswil, Switzerland; [email protected] (J.B.); [email protected] (A.P.) * Correspondence: [email protected]; Tel.: +41-58-934-5438 These authors contributed equally. y Received: 15 November 2019; Accepted: 2 December 2019; Published: 5 December 2019 Abstract: The introduction of a halogen atom into a small molecule can effectively modulate its properties, yielding bioactive substances of agrochemical and pharmaceutical interest. Consequently, the development of selective halogenation strategies is of high technological value. Besides chemical methodologies, enzymatic halogenations have received increased interest as they allow the selective installation of halogen atoms in molecular scaffolds of varying complexity under mild reaction conditions. Today, a comprehensive library of aromatic halogenases exists, and enzyme as well as reaction engineering approaches are being explored to broaden this enzyme family’s biocatalytic application range. In this review, we highlight recent developments in the sourcing, engineering, and application of flavin-dependent halogenases with a special focus on chemoenzymatic and coupled biosynthetic approaches. Keywords: flavin-dependent halogenases; enzyme engineering; anion promiscuity; reaction engineering; co-factor recycling; biocatalysis 1. Introduction In nature, over 5000 halogenated products have been described since their first discovery over 100 years ago including potent antimicrobial agents such as vancomycin and chloramphenicol (Figure1)[ 1]. The incorporation of a halogen can significantly alter a molecule’s properties and may impact its bioactivity, metabolism, and pharmacokinetic profile [2]. As a consequence, the selective halogenation of small molecules is of great interest in the pharmaceutical and agrochemical industry. Around 20% of small molecule drugs and approximately 30% of agrochemicals are halogenated [3] Examples in the pharmaceutical industry include blockbusters such as rivaroxaban and empagliflozin with annual sales revenues in 2018 of $6.6 and $2.3 billion, respectively (Figure1)[ 4]. Furthermore, C-X motifs are useful synthetic handles for modification of the molecule in further chemical steps, making halogenated species common intermediates in synthetic manufacturing routes [5,6]. However, chemical halogenation, even though being a well-established technology, suffers from the use of hazardous or even highly toxic chemicals and sometimes poor atom efficiency [7,8]. In addition, chemical methods often lack selectivity or have specific demands on the substrate structure [8,9]. Consequently, the replacement of chemical halogenation by biological means may offer a more selective, greener, and less dangerous process route toward molecules of industrial interest. Biocatalytic halogenation is carried out by enzymes called halogenases or haloperoxidases, which can be classified according to their catalytic mechanism: heme, vanadium, and flavin-dependent halogenases follow an electrophilic mechanism, while non-heme iron halogenases halogenate through the formation of radical intermediates, and S-adenosyl-L-methionine (SAM) fluorinases react via a Catalysts 2019, 9, 1030; doi:10.3390/catal9121030 www.mdpi.com/journal/catalysts Catalysts 2019, 9, 1030 2 of 20 nucleophilic pathway [10]. This review will focus on work carried out on flavin-dependent halogenases (electrophilic halogenases), which in contrast to heme- and vanadium-dependent halogenases, Catalysts 2019, 9, 1030 2 of 21 selectively derivatize small molecules, making them especially interesting for applications. Figure 1. SelectionFigure of 1. valuableSelection of halogenated valuable halogenated agrochem agrochemicals,icals, pharmaceuticals pharmaceuticals and natural and products. natural products. Flavin-dependent halogenases (Fl-Hals) use reduced flavin, bound in the FAD binding site of the Biocatalyticenzyme, halogenation to create a C4a-hydroperoxyflavin is carried out speciesby en uponzymes reaction called with oxygenhalogenases [11]. The generated or haloperoxidases, peroxy species cannot react directly with the substrate as the flavin binding site and the substrate which can bebinding classified site are separatedaccordin byg an approximatelyto their catalytic 10 Å long tunnelmechanism: [12–15]. Instead, heme, crystal vanadium, structures of and flavin- dependent halogenasesflavin-dependent follow halogenases an showelectrophilic the presence ofmechanism, a chloride binding while site near non-heme the flavin binding iron halogenases halogenate throughsite [15,16 the], and formation mechanistic studies of suggestradical that in a nucleophilictermediates, attack ofand the chlorideS-adenosyl-L-methionine on the peroxyflavin (SAM) may create hypohalous acid (HOX) [11]. The hypohalous acid then travels to the substrate binding fluorinases reactsite via and a is nucleophilic believed to interact pathway with an active [10]. site This lysine review either by will reaction focus or via on hydrogen work carried bonding out on flavin- dependent halogenases(Figure2)[ 16, 17(electrophilic]. It is assumed that halogenases), this electrophilic chlorine which species in iscontrast responsible to for heme- electrophilic and vanadium- aromatic substitution of the proximally bound substrates. Notably, an active site glutamate residue dependent halogenases,has also been foundselectively important derivatize for catalysis; however,small molecules, its precise role inmaking the reaction them mechanism especially still interesting for applications.remains debated [2,18]. Flavin-dependent halogenases (Fl-Hals) use reduced flavin, bound in the FAD binding site of the enzyme, to create a C4a-hydroperoxyflavin species upon reaction with oxygen [11]. The generated peroxy species cannot react directly with the substrate as the flavin binding site and the substrate binding site are separated by an approximately 10 Å long tunnel [12–15]. Instead, crystal structures of flavin-dependent halogenases show the presence of a chloride binding site near the flavin binding site [15,16], and mechanistic studies suggest that a nucleophilic attack of the chloride on the peroxyflavin may create hypohalous acid (HOX) [11]. The hypohalous acid then travels to the substrate binding site and is believed to interact with an active site lysine either by reaction or via hydrogen bonding (Figure 2) [16,17]. It is assumed that this electrophilic chlorine species is responsible for electrophilic aromatic substitution of the proximally bound substrates. Notably, an active site glutamate residue has also been found important for catalysis; however, its precise role in the reaction mechanism still remains debated [2,18]. Catalysts 2019, 9, 1030 3 of 20 Catalysts 2019, 9, 1030 3 of 21 FigureFigure 2. Reaction 2. Reaction mechanism mechanism of flavin-dependent of flavin-dependent halo halogenases.genases. Reduced Reduced flavin flavin reacts reacts with with oxygen oxygen to formto form a peroxyflavin a peroxyflavin species, species, which which in turn in turn reac reactsts with with a halide a halide ion, ion, generating generating free free hypohalous hypohalous acid.acid. The Thehypohalous hypohalous acid acid travels travels through through an a anpproximately approximately 10 Å 10 tunnel Å tunnel before before reacting reacting with with an an activeactive site site lysine. lysine. Through Through an electrophilicelectrophilic aromatic aromatic substitution substitution mechanism, mechanism, the halogenatedthe halogenated aromatic aromaticcompound compound is formed. is formed. 2. Enzyme2. Enzyme Diversity Diversity and and Substrate Substrate Scope Scope To Todate, date, a plethora a plethora of flavin-dependent of flavin-dependent halogenase halogenasess (Fl-Hals) (Fl-Hals) has has been been identified. identified. Fl-Hals Fl-Hals accept accept a a rangerange of didifferentfferent substratessubstrates but but preferentially preferentially have have been been found found to halogenate to halogenate L-tryptophan L-tryptophan (Figure 3: (Figurered square) 3: red [19square)], pyrrole-like [19], pyrrole-like compounds (Figurecompou3:nds green (Figure triangle) 3: [ green20], phenolic triangle) compounds [20], phenolic (Figure 3: compoundsblue circle) (Figure [21], and 3: blue indole circle) (Figure [21],3: and orange indole square) (Figure [ 22 ].3: Inorange this section, square) we [22]. will In give this ansection, overview we of willthe give natural an overview diversity of flavin-dependentthe natural diversit halogenasesy of flavin-dependent emphasizing halogenases the recently discoveredemphasizing members the recentlyof this discovered enzyme family. members of this enzyme family. TheThe first first flavin-dependent flavin-dependent halogenaseshalogenases were were identified identified in thein mid-1990sthe mid-1990s [19,23]. [19,23]. PrnA, discovered PrnA, discoveredin Pseudomonas in Pseudomonas fluorescens influorescens 1998, is for in example 1998, involvedis for example in the biosynthesis involved ofin pyrrolnitrin,the biosynthesis a secondary of pyrrolnitrin,metabolite a secondary derived from metabolite L-tryptophan derived with from a L-tryptophan strong antifungal with