Protein Evolution and Design.

This article introduces the Protein Evolution and Design theme of the Annual Review of Biochemistry Volume 87.

Extended Biocatalytic Halogenation Cascades Involving a Single-Polypeptide Regeneration System for Diffusible FADH2.

Flavin-dependent halogenases have attracted increasing interest for aryl halogenation at unactivated C-H positions because they are characterised by high regioselectivity, while requiring only FADH2 , halide salts, and O2 . Their use in combined crosslinked enzyme aggregates (combiCLEAs) together with an NADH-dependent flavin reductase and an NADH-regeneration system for the preparative halogenation of tryptophan and indole derivatives has been previously described. However, multiple cultivations and protein purification steps are necessary for their production. We present a bifunctional regeneration enzyme for two-step catalytic flavin regeneration using phosphite as an inexpensive sacrificial substrate. This fusion protein proved amenable to co-expression with various flavin-dependent Trp-halogenases and enables carrier-free immobilisation as combiCLEAs from a single cultivation for protein production and the preparative synthesis of halotryptophan. The scalability of this system was demonstrated by fed-batch fermentation in bench-top bioreactors on a 2.5 L scale. Furthermore, the inclusion of a 6-halotryptophan-specific dioxygenase into the co-expression strain further converts the halogenation product to the kynurenine derivative. This reaction cascade enables the one-pot synthesis of l-4-Cl-kynurenine and its brominated analogue on a preparative scale.

Colibrimycins, Novel Halogenated Hybrid Polyketide Synthase-Nonribosomal Peptide Synthetase (PKS-NRPS) Compounds Produced by Streptomyces sp. Strain CS147.

The improvement of genome sequencing techniques has brought to light the biosynthetic potential of actinomycetes due to the large number of gene clusters they present compared to the number of known compounds. Genome mining is a recent strategy in the search for novel bioactive compounds, which involves the analysis of sequenced genomes to identify uncharacterized natural product biosynthetic gene clusters, many of which are cryptic or silent under laboratory conditions, and to develop experimental approaches to identify their products. Owing to the importance of halogenation in terms of structural diversity, bioavailability, and bioactivity, searching for new halogenated bioactive compounds has become an interesting issue in the field of natural product discovery. Following this purpose, a screening for halogenase coding genes was performed on 12 Streptomyces strains isolated from fungus-growing ants of the Attini tribe. Using the bioinformatics tools antiSMASH and BLAST, six halogenase coding genes were identified. Some of these genes were located within biosynthetic gene clusters (BGCs), which were studied by construction of several mutants for the identification of the putative halogenated compounds produced. The comparison of the metabolite production profile of wild-type strains and their corresponding mutants by ultrahigh-performance liquid chromatography-UV and high-performance liquid chromatography-mass spectrometry allowed us the identification of a novel family of halogenated compounds in Streptomyces sp. strain CS147, designated colibrimycins. IMPORTANCE Genome mining has proven its usefulness in the search for novel bioactive compounds produced by microorganisms, and halogenases comprise an interesting starting point. In this work, we have identified a new halogenase coding gene that led to the discovery of novel lipopetide nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS)-derived natural products, the colibrimycins, produced by Streptomyces sp. strain CS147, isolated from the Attini ant niche. Some colibrimycins display an unusual α-ketoamide moiety in the peptide structure. Although its biosynthetic origin remains unknown, its presence might be related to a hypothetical inhibition of virus proteases, and, together with the presence of the halogenase, it represents a feature to be incorporated in the arsenal of structural modifications available for combinatorial biosynthesis.

Halogenation in Fungi: What Do We Know and What Remains to Be Discovered?

In nature, living organisms produce a wide variety of specialized metabolites to perform many biological functions. Among these specialized metabolites, some carry halogen atoms on their structure, which can modify their chemical characteristics. Research into this type of molecule has focused on how organisms incorporate these atoms into specialized metabolites. Several families of enzymes have been described gathering metalloenzymes, flavoproteins, or S-adenosyl-L-methionine (SAM) enzymes that can incorporate these atoms into different types of chemical structures. However, even though the first halogenation enzyme was discovered in a fungus, this clade is still lagging behind other clades such as bacteria, where many enzymes have been discovered. This review will therefore focus on all halogenation enzymes that have been described in fungi and their associated metabolites by searching for proteins available in databases, but also by using all the available fungal genomes. In the second part of the review, the chemical diversity of halogenated molecules found in fungi will be discussed. This will allow the highlighting of halogenation mechanisms that are still unknown today, therefore, highlighting potentially new unknown halogenation enzymes.

Distribution and diversity of dimetal-carboxylate halogenases in cyanobacteria.

BACKGROUND: Halogenation is a recurring feature in natural products, especially those from marine organisms. The selectivity with which halogenating enzymes act on their substrates renders halogenases interesting targets for biocatalyst development. Recently, CylC - the first predicted dimetal-carboxylate halogenase to be characterized - was shown to regio- and stereoselectively install a chlorine atom onto an unactivated carbon center during cylindrocyclophane biosynthesis. Homologs of CylC are also found in other characterized cyanobacterial secondary metabolite biosynthetic gene clusters. Due to its novelty in biological catalysis, selectivity and ability to perform C-H activation, this halogenase class is of considerable fundamental and applied interest. The study of CylC-like enzymes will provide insights into substrate scope, mechanism and catalytic partners, and will also enable engineering these biocatalysts for similar or additional C-H activating functions. Still, little is known regarding the diversity and distribution of these enzymes. RESULTS: In this study, we used both genome mining and PCR-based screening to explore the genetic diversity of CylC homologs and their distribution in bacteria. While we found non-cyanobacterial homologs of these enzymes to be rare, we identified a large number of genes encoding CylC-like enzymes in publicly available cyanobacterial genomes and in our in-house culture collection of cyanobacteria. Genes encoding CylC homologs are widely distributed throughout the cyanobacterial tree of life, within biosynthetic gene clusters of distinct architectures (combination of unique gene groups). These enzymes are found in a variety of biosynthetic contexts, which include fatty-acid activating enzymes, type I or type III polyketide synthases, dialkylresorcinol-generating enzymes, monooxygenases or Rieske proteins. Our study also reveals that dimetal-carboxylate halogenases are among the most abundant types of halogenating enzymes in the phylum Cyanobacteria. CONCLUSIONS: Our data show that dimetal-carboxylate halogenases are widely distributed throughout the Cyanobacteria phylum and that BGCs encoding CylC homologs are diverse and mostly uncharacterized. This work will help guide the search for new halogenating biocatalysts and natural product scaffolds.

Biosynthetic Machinery of 6-Hydroxymellein Derivatives Leading to Cyclohelminthols and Palmaenones.

Oxygenated cyclopentene systems are unique structural motifs found in fungal polyketides such as terrein, cyclohelminthols, and palmaenones. Here we report the identification of the biosynthetic gene clusters for cyclohelminthols and palmaenones and the functional characterization of the polyketide synthases and halogenases involved in the construction of 6-hydroxymellein derivatives. Heterologous expression in Aspergillus oryzae demonstrated that 6-hydroxymellein is a common biosynthetic intermediate and that chlorination occurs in the early stages of its products' biosynthesis. This was further confirmed by in vitro enzymatic reactions conducted in the presence of recombinant proteins. Plausible means of biogenesis of fungal polyketides from 6-hydroxymellein derivatives, additionally supported by the reported labeling patterns of terrein and structurally related fungal polyketides, are also discussed. This study sets the stage for elucidation of the biosynthetic machinery of fungal polyketides of this type.

Characterization of a Tryptophan 6-Halogenase from Streptomyces albus and Its Regioselectivity Determinants.

Tryptophan halogenases are found in diverse organisms and catalyze regiospecific halogenation. They play an important role in the biosynthesis of halogenated indole alkaloids, which are biologically active and of therapeutic importance. Here, a tryptophan 6-halogenase (SatH) from Streptomyces albus was characterized by using a whole-cell reaction system in Escherichia coli. SatH showed substrate specificity for chloride and bromide ions, leading to regiospecific halogenation at the C6-position of l-tryptophan. In addition, SatH exhibited higher performance in bromination than that of previously reported tryptophan halogenases in the whole-cell reaction system. Through structure-based protein mutagenesis, it has been revealed that two consecutive residues, A78/V79 in SatH and G77/I78 in PyrH, are key determinants in the regioselectivity difference between tryptophan 6- and 5-halogenases. Substituting the AV with GI residues switched the regioselectivity of SatH by moving the orientation of tryptophan. These data contribute to an understanding of the key residues that determine the regioselectivity of tryptophan halogenases.

Flavin Adenine Dinucleotide-Dependent Halogenase XanH and Engineering of Multifunctional Fusion Halogenases.

Xantholipin (compound 1), a polycyclic xanthone antibiotic, exhibited strong antibacterial activities and showed potent cytotoxicity. The biosynthetic gene cluster of compound 1 has been identified in our previous work, and the construction of xanthone nucleus has been well demonstrated. However, limited information of the halogenation involved in compound 1 biosynthesis is available. In this study, based on the genetic manipulation and biochemical assay, we characterized XanH as an indispensable flavin adenine dinucleotide (FAD)-dependent halogenase (FDH) for the biosynthesis of compound 1. XanH was found to be a bifunctional protein capable of flavin reduction and chlorination and exclusively used the NADH. However, the reduced flavin could not be fully and effectively utilized, and the presence of an extra flavin reductase (FDR) and chemical-reducing agent could promote the halogenation. XanH accepted its natural free-standing substrate with angular fused polycyclic aromatic systems. Meanwhile, it exhibited moderate halogenation activity and possessed high substrate specificity. The requirement of extra FDR for higher halogenation activity is tedious for future engineering. To facilitate efforts in engineering XanH derivative proteins, we constructed the self-sufficient FDR-XanH fusion proteins. The fusion protein E1 with comparable activities to that of XanH could be used as a good alternative for future protein engineering. Taken together, these findings reported here not only improve the understanding of polycyclic xanthones biosynthesis but also expand the substrate scope of FDH and pave the way for future engineering of biocatalysts for new active substance synthesis.IMPORTANCE Halogenation is important in medicinal chemistry and plays an essential role in the biosynthesis of active secondary metabolites. Halogenases have evolved to catalyze reactions with high efficiency and selectivity, and engineering efforts have been made to engage the selective reactivity in natural product biosynthesis. The enzymatic halogenations are an environmentally friendly approach with high regio- and stereoselectivity, which make it a potential complement to organic synthesis. FDHs constitute one of the most extensively elucidated class of halogenases; however, the inventory awaits to be expanded for biotechnology applications and for the generation of halogenated natural product analogues. In this study, XanH was found to reduce flavin and halogenated the freely diffusing natural substrate with an angular fused hexacyclic scaffold, findings which were different from those for the exclusively studied FDHs. Moreover, the FDR-XanH fusion protein E1 with comparable reactivity to that of XanH serves as a successful example of genetic fusions and sets an important stage for future protein engineering.

Evolved Aliphatic Halogenases Enable Regiocomplementary C-H Functionalization of a Pharmaceutically Relevant Compound.

Non-heme iron halogenases are synthetically valuable biocatalysts that are capable of halogenating unactivated sp3 -hybridized carbon centers with high stereo- and regioselectivity. The reported substrate scope of these enzymes, however, is limited primarily to the natural substrates and their analogues. We engineered the halogenase WelO5* for chlorination of a martinelline-derived fragment. Using structure-guided evolution, a halogenase variant with a more than 290-fold higher total turnover number and a 400-fold higher apparent kcat compared to the wildtype enzyme was generated. Moreover, we identified key positions in the active site that allow direction of the halogen to different positions in the target substrate. This is the first example of enzyme engineering to expand the substrate scope of a non-heme iron halogenase beyond the native indole-alkaloid-type substrates. The highly evolvable nature of WelO5* underscores the usefulness of this enzyme family for late-stage halogenation.

Microbial and Functional Biodiversity Patterns in Sponges that Accumulate Bromopyrrole Alkaloids Suggest Horizontal Gene Transfer of Halogenase Genes.

Marine sponge holobionts harbor complex microbial communities whose members may be the true producers of secondary metabolites accumulated by sponges. Bromopyrrole alkaloids constitute a typical class of secondary metabolites isolated from sponges that very often display biological activities. Bromine incorporation into secondary metabolites can be catalyzed by either halogenases or haloperoxidases. The diversity of the metagenomes of sponge holobiont species containing bromopyrrole alkaloids (Agelas spp. and Tedania brasiliensis) as well as holobionts devoid of bromopyrrole alkaloids spanning in a vast biogeographic region (approx. Seven thousand km) was studied. The origin and specificity of the detected halogenases was also investigated. The holobionts Agelas spp. and T. brasiliensis did not share microbial halogenases, suggesting a species-specific pattern. Bacteria of diverse phylogenetic origins encoding halogenase genes were found to be more abundant in bromopyrrole-containing sponges. The sponge holobionts (e.g., Agelas spp.) with the greatest number of sequences related to clustered, interspaced, short, palindromic repeats (CRISPRs) exhibited the fewest phage halogenases, suggesting a possible mechanism of protection from phage infection by the sponge host. This study highlights the potential of phages to transport halogenases horizontally across host sponges, particularly in more permissive holobiont hosts, such as Tedania spp.

Dioxygen activation by nonheme iron enzymes with the 2-His-1-carboxylate facial triad that generate high-valent oxoiron oxidants.

The 2-His-1-carboxylate facial triad is a widely used scaffold to bind the iron center in mononuclear nonheme iron enzymes for activating dioxygen in a variety of oxidative transformations of metabolic significance. Since the 1990s, over a hundred different iron enzymes have been identified to use this platform. This structural motif consists of two histidines and the side chain carboxylate of an aspartate or a glutamate arranged in a facial array that binds iron(II) at the active site. This triad occupies one face of an iron-centered octahedron and makes the opposite face available for the coordination of O2 and, in many cases, substrate, allowing the tailoring of the iron-dioxygen chemistry to carry out a plethora of diverse reactions. Activated dioxygen-derived species involved in the enzyme mechanisms include iron(III)-superoxo, iron(III)-peroxo, and high-valent iron(IV)-oxo intermediates. In this article, we highlight the major crystallographic, spectroscopic, and mechanistic advances of the past 20 years that have significantly enhanced our understanding of the mechanisms of O2 activation and the key roles played by iron-based oxidants.

RadH: A Versatile Halogenase for Integration into Synthetic Pathways.

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.

Discovery of Unusual Biaryl Polyketides by Activation of a Silent Streptomyces venezuelae Biosynthetic Gene Cluster.

Comparative transcriptional profiling of a ΔbldM mutant of Streptomyces venezuelae with its unmodified progenitor revealed that the expression of a cryptic biosynthetic gene cluster containing both type I and type III polyketide synthase genes is activated in the mutant. The 29.5 kb gene cluster, which was predicted to encode an unusual biaryl metabolite, which we named venemycin, and potentially halogenated derivatives, contains 16 genes including one-vemR-that encodes a transcriptional activator of the large ATP-binding LuxR-like (LAL) family. Constitutive expression of vemR in the ΔbldM mutant led to the production of sufficient venemycin for structural characterisation, confirming its unusual biaryl structure. Co-expression of the venemycin biosynthetic gene cluster and vemR in the heterologous host Streptomyces coelicolor also resulted in venemycin production. Although the gene cluster encodes two halogenases and a flavin reductase, constitutive expression of all three genes led to the accumulation only of a monohalogenated venemycin derivative, both in the native producer and the heterologous host. A competition experiment in which equimolar quantities of sodium chloride and sodium bromide were fed to the venemycin-producing strains resulted in the preferential incorporation of bromine, thus suggesting that bromide is the preferred substrate for one or both halogenases.

Aliphatic Halogenase Enables Late-Stage C-H Functionalization: Selective Synthesis of a Brominated Fischerindole Alkaloid with Enhanced Antibacterial Activity.

The anion promiscuity of a newly discovered standalone aliphatic halogenase WelO5 was probed and enabled the selective synthesis of 13R-bromo-12-epi-fischerindole U via late-stage enzymatic functionalization of an unactivated sp(3) C-H bond. Pre-saturating the WelO5 active site with a non-native bromide anion was found to be critical to the highly selective in vitro transfer of bromine, instead of chlorine, to the target carbon center and also allowed the relative binding affinity of bromide and chloride towards the WelO5 enzyme to be assessed. This study further revealed the critical importance of halogen substitution on modulating the antibiotic activity of fischerindole alkaloids and highlights the promise of WelO5-type aliphatic halogenases as enzymatic tools to fine-tune the bioactivity of complex natural products.

Specific Enzymatic Halogenation-From the Discovery of Halogenated Enzymes to Their Applications In†Vitro and In†Vivo.

During the last 20 years, focus has shifted from haloperoxidases to flavin-dependent and non-heme-iron halogenases because of their proven involvement in the biosynthesis of halogenated metabolites in different organisms and the regioselectivity of their reactions. During the first 10-12 years, the main research topics were the detection of halogenases as well as the elucidation of three-dimensional structures and reaction mechanisms. This Review mainly deals with studies on halogenating enzymes published between 2010 and 2015. It focusses on the elucidation of the involvement of halogenating enzymes in halometabolite biosynthesis, application of halogenases in in vivo and in vitro systems, in vivo modification of biosynthetic pathways in bacteria and plants, improvement of enzyme stability, broadening of substrate specificity, and the combination of biocatalysis with chemical synthesis to produce new compounds.

A High-Throughput Fluorescence Assay to Determine the Activity of Tryptophan Halogenases.

Biocatalytic halogenation with tryptophan halogenases is hampered by severe limitations such as low activity and stability. These drawbacks can be overcome by directed evolution, but for screening large mutant libraries, a facile high-throughput method is required. Therefore, we developed a quantitative halogenase assay based on a Suzuki-Miyaura cross-coupling towards the formation of a fluorescent aryltryptophan. The technique was optimized for application in crude E. coli lysate without intermediary purification steps, and was used for quantitatively monitoring the formation of halogenated tryptophans with high specificity by facile fluorescence screening in microtiter plates. This novel screening approach was exploited to engineer a thermostable tryptophan 6-halogenase. Libraries were constructed by error-prone PCR and selected for improved thermal resistance simply by fluorogenic cross-coupling. Our method led to an enzyme variant with substantially increased thermal stability and 2.5-fold improved activity.

Halogenase Engineering for the Generation of New Natural Product Analogues.

Halogenases catalyze the incorporation of halogen atoms into organic molecules. Given the importance that halogenation has on the biological activity of small molecules, these enzymes have been subjected to intense engineering efforts to make them more suitable for biotechnology applications. The ability to biohalogenate complex molecules provides, in principle, the opportunity for rapid generation of a series of analogues with new or improved properties. Here we discuss the potential and limitations of using halogenases as biocatalysts, including recent advances in engineering halogenases to generate halogenated natural product analogues.

Enzymatic halogenation of tryptophan on a gram scale.

Halogenated arenes are important building blocks in medicinal and agrochemistry. Chemical electrophilic aromatic halogenation requires molecular halogen, whereas FAD-dependent halogenases form halogenated arenes with high regioselectivity while only halide salts and O2 are required. This reaction proceeds at room temperature in aqueous media. However, enzymatic halogenation is considered inefficient, mainly because halogenases are not stable. Thus, the preparative application remained elusive. We were able to show that the long-term stability and, hence, the preparative efficiency of the tryptophan-7-halogenase RebH can be significantly improved by immobilization together with the other enzymes required for cofactor regeneration. We established a facile scalable method suitable for the halogenation of tryptophan and its derivatives on a gram scale using a solid, multifunctional, and recyclable biocatalyst; this immobilization strategy might also be applicable for other FAD-dependent halogenases.

Improving the stability and catalyst lifetime of the halogenase RebH by directed evolution.

We previously reported that the halogenase RebH catalyzes selective halogenation of several heterocycles and carbocycles, but product yields were limited by enzyme instability. Here, we use directed evolution to engineer an RebH variant, 3-LR, with a Topt over 5 °C higher than that of wild-type, and 3-LSR, with a Tm 18 °C higher than that of wild-type. These enzymes provided significantly improved conversion (up to fourfold) for halogenation of tryptophan and several non-natural substrates. This initial evolution of RebH not only provides improved enzymes for immediate synthetic applications, but also establishes a robust protocol for further halogenase evolution.

A tryptophan 6-halogenase and an amidotransferase are involved in thienodolin biosynthesis.

The biosynthetic gene cluster for the plant growth-regulating compound thienodolin was identified in and cloned from the producer organism Streptomyces albogriseolus MJ286-76F7. Sequence analysis of a 27 kb DNA region revealed the presence of 21 ORFs, 14 of which are involved in thienodolin biosynthesis. Three insertional inactivation mutants were generated in the sequenced region to analyze their involvement in thienodolin biosynthesis and to functionally characterize specific genes. The gene inactivation experiments together with enzyme assays with enzymes obtained by heterologous expression and feeding studies showed that the first step in thienodolin biosynthesis is catalyzed by a tryptophan 6-halogenase and that the last step is the formation of a carboxylic amide group catalyzed by an amidotransferase. The results led to a hypothetical model for thienodolin biosynthesis.