Polar Interactions between Substrate and Flavin Control Iodotyrosine Deiodinase Function.

Flavin cofactors offer a wide range of chemical mechanisms to support a great diversity in catalytic function. As a corollary, such diversity necessitates careful control within each flavoprotein to limit its function to an appropriate subset of possible reactions and substrates. This task falls to the protein environment surrounding the flavin in most enzymes. For iodotyrosine deiodinase that catalyzes a reductive dehalogenation of halotyrosines, substrates can dictate the chemistry available to the flavin. Their ability to stabilize the necessary one-electron reduced semiquinone form of flavin strictly depends on a direct coordination between the flavin and α-ammonium and carboxylate groups of its substrates. While perturbations to the carboxylate group do not significantly affect binding to the resting oxidized form of the deiodinase, dehalogenation (kcat/Km) is suppressed by over 2000-fold. Lack of the α-ammonium group abolishes detectable binding and dehalogenation. Substitution of the ammonium group with a hydroxyl group does not restore measurable binding but does support dehalogenation with an efficiency greater than those of the carboxylate derivatives. Consistent with these observations, the flavin semiquinone does not accumulate during redox titration in the presence of inert substrate analogues lacking either the α-ammonium or carboxylate groups. As a complement, a nitroreductase activity based on hydride transfer is revealed for the appropriate substrates with perturbations to their zwitterion.

Methods for biochemical characterization of flavin-dependent N-monooxygenases involved in siderophore biosynthesis.

Siderophores are essential molecules released by some bacteria and fungi in iron-limiting environments to sequester ferric iron, satisfying metabolic needs. Flavin-dependent N-hydroxylating monooxygenases (NMOs) catalyze the hydroxylation of nitrogen atoms to generate important siderophore functional groups such as hydroxamates. It has been demonstrated that the function of NMOs is essential for virulence, implicating these enzymes as potential drug targets. This chapter aims to serve as a resource for the characterization of NMO's enzymatic activities using several biochemical techniques. We describe assays that allow for the determination of steady-state kinetic parameters, detection of hydroxylated amine products, measurement of the rate-limiting step(s), and the application toward drug discovery efforts. While not exhaustive, this chapter will provide a foundation for the characterization of enzymes involved in siderophore biosynthesis, allowing for gaps in knowledge within the field to be addressed.

Asymmetric Sulfoxidations Catalyzed by Bacterial Flavin-Containing Monooxygenases.

Flavin-containing monooxygenase from Methylophaga sp. (mFMO) was previously discovered to be a valuable biocatalyst used to convert small amines, such as trimethylamine, and various indoles. As FMOs are also known to act on sulfides, we explored mFMO and some mutants thereof for their ability to convert prochiral aromatic sulfides. We included a newly identified thermostable FMO obtained from the bacterium Nitrincola lacisaponensis (NiFMO). The FMOs were found to be active with most tested sulfides, forming chiral sulfoxides with moderate-to-high enantioselectivity. Each enzyme variant exhibited a different enantioselective behavior. This shows that small changes in the substrate binding pocket of mFMO influence selectivity, representing a tunable biocatalyst for enantioselective sulfoxidations.

A Redox-Reversible Switch of DNA Hydrogen Bonding and Structure.

Modulating molecular structure and function at the nanoscale drives innovation across wide-ranging technologies. Electrical control of the bonding of individual DNA base pairs endows DNA with precise nanoscale structural reconfigurability, benefiting efforts in DNA origami and actuation. Here, alloxazine DNA base surrogates were synthesized and incorporated into DNA duplexes to function as a redox-active switch of hydrogen bonding. Circular dichroism (CD) revealed that 24-mer DNA duplexes containing one or two alloxazines exhibited CD spectra and melting transitions similar to DNA with only canonical bases, indicating that the constructs adopt a B-form conformation. However, duplexes were not formed when four or more alloxazines were incorporated into a 24-mer strand. Thiolated duplexes incorporating alloxazines were self-assembled onto multiplexed gold electrodes and probed electrochemically. Square-wave voltammetry (SWV) revealed a substantial reduction peak centered at -0.272 V vs Ag/AgCl reference. Alternating between alloxazine oxidizing and reducing conditions modulated the SWV peak in a manner consistent with the formation and loss of hydrogen bonding, which disrupts the base pair stacking and redox efficiency of the DNA construct. These alternating signals support the assertion that alloxazine can function as a redox-active switch of hydrogen bonding, useful in controlling DNA and bioinspired assemblies.

Versatile roles of protein flavinylation in bacterial extracyotosolic electron transfer.

Bacteria perform diverse redox chemistries in the periplasm, cell wall, and extracellular space. Electron transfer for these extracytosolic activities is frequently mediated by proteins with covalently bound flavins, which are attached through post-translational flavinylation by the enzyme ApbE. Despite the significance of protein flavinylation to bacterial physiology, the basis and function of this modification remain unresolved. Here we apply genomic context analyses, computational structural biology, and biochemical studies to address the role of ApbE flavinylation throughout bacterial life. We identify ApbE flavinylation sites within structurally diverse protein domains and show that multi-flavinylated proteins, which may mediate longer distance electron transfer via multiple flavinylation sites, exhibit substantial structural heterogeneity. We identify two novel classes of flavinylation substrates that are related to characterized proteins with non-covalently bound flavins, providing evidence that protein flavinylation can evolve from a non-covalent flavoprotein precursor. We further find a group of structurally related flavinylation-associated cytochromes, including those with the domain of unknown function DUF4405, that presumably mediate electron transfer in the cytoplasmic membrane. DUF4405 homologs are widespread in bacteria and related to ferrosome iron storage organelle proteins that may facilitate iron redox cycling within ferrosomes. These studies reveal a complex basis for flavinylated electron transfer and highlight the discovery power of coupling comparative genomic analyses with high-quality structural models. IMPORTANCE: This study explores the mechanisms bacteria use to transfer electrons outside the cytosol, a fundamental process involved in energy metabolism and environmental interactions. Central to this process is a phenomenon known as flavinylation, where a flavin molecule-a compound related to vitamin B2-is covalently attached to proteins, to enable electron transfer. We employed advanced genomic analysis and computational modeling to explore how this modification occurs across different bacterial species. Our findings uncover new types of proteins that undergo this modification and highlight the diversity and complexity of bacterial electron transfer mechanisms. This research broadens our understanding of bacterial physiology and informs potential biotechnological applications that rely on microbial electron transfer, including bioenergy production and bioremediation.

Characterization of a deazaflavin analog as a potent inhibitor of multidrug resistance-associated protein 1.

Selective inhibition of overexpressed ATP binding cassette (ABC) transporters is an attractive approach to enhancing the efficacy of chemotherapeutics in multidrug resistant cancers. Previously, we reported that the cancer sensitizing effect of deazaflavin analogs, an important chemotype for developing combination treatments with topoisomerase II (TOP2) poisons, is associated with increased intracellular drug accumulation. Here we report the characterization of ZW-1226, a deazaflavin analog, as a potent inhibitor of multidrug resistance-associated protein 1 (MRP1). Specifically, ZW-1226 inhibited MRP1 with a 16-fold higher potency than the most widely used positive control MK-571 in vesicular transport assay and displayed excellent selectivity indices exceeding 100 over other major ABC transporters, including P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), MRP2 and MRP3. Mechanistically, we revealed that its MRP1 inhibitory action requires the participation of GSH. In chemo-sensitization test, ZW-1226 fully reversed the MRP1-mediated drug resistance to TOP2 poisons etoposide (ETP) and doxorubicin (DOX) in H69AR cells and conferred CC50s comparable to those in the sensitive parental NCI-H69 cells. The sensitization was associated with boosted intracellular accumulation of ETP and DOX and elevated endogenous GSH. Moreover, ZW-1226 showed potential to occupy the leukotriene C4 binding site in molecular docking with bovine MRP1, presumably with the help of GSH. Lastly, ZW-1226 exhibited high tissue to plasma partitions in mice but did not alter ETP distribution to normal tissues, suggesting it could be a viable lead with desirable pharmacokinetic properties to warrant further investigation.

Exploring the impact of flavin homeostasis on cancer cell metabolism.

Flavins and their associated proteins have recently emerged as compelling players in the landscape of cancer biology. Flavins, encompassing flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), serve as coenzymes in a multitude of cellular processes, such as metabolism, apoptosis, and cell proliferation. Their involvement in oxidative phosphorylation, redox homeostasis, and enzymatic reactions has long been recognized. However, recent research has unveiled an extended role for flavins in the context of cancer. In parallel, riboflavin transporters (RFVTs), FAD synthase (FADS), and riboflavin kinase (RFK) have gained prominence in cancer research. These proteins, responsible for riboflavin uptake, FAD biosynthesis, and FMN generation, are integral components of the cellular machinery that governs flavin homeostasis. Dysregulation in the expression/function of these proteins has been associated with various cancers, underscoring their potential as diagnostic markers, therapeutic targets, and key determinants of cancer cell behavior. This review embarks on a comprehensive exploration of the multifaceted role of flavins and of the flavoproteins involved in nucleus-mitochondria crosstalk in cancer. We journey through the influence of flavins on cancer cell energetics, the modulation of RFVTs in malignant transformation, the diagnostic and prognostic significance of FADS, and the implications of RFK in drug resistance and apoptosis. This review also underscores the potential of these molecules and processes as targets for novel diagnostic and therapeutic strategies, offering new avenues for the battle against this relentless disease.

Identifying and Engineering Flavin Dependent Halogenases for Selective Biocatalysis.

ConspectusOrganohalogen compounds are extensively used as building blocks, intermediates, pharmaceuticals, and agrochemicals due to their unique chemical and biological properties. Installing halogen substituents, however, frequently requires functionalized starting materials and multistep functional group interconversion. Several classes of halogenases evolved in nature to enable halogenation of a different classes of substrates; for example, site-selective halogenation of electron rich aromatic compounds is catalyzed by flavin-dependent halogenases (FDHs). Mechanistic studies have shown that these enzymes use FADH2 to reduce O2 to water with concomitant oxidation of X- to HOX (X = Cl, Br, I). This species travels through a tunnel within the enzyme to access the FDH active site. Here, it is believed to interact with an active site lysine proximal to bound substrate, enabling electrophilic halogenation with selectivity imparted via molecular recognition, rather than directing groups or strong electronic activation.The unique selectivity of FDHs led to several early biocatalysis efforts, preparative halogenation was rare, and the hallmark catalyst-controlled selectivity of FDHs did not translate to non-native substrates. FDH engineering was limited to site-directed mutagenesis, which resulted in modest changes in site-selectivity or substrate preference. To address these limitations, we optimized expression conditions for the FDH RebH and its cognate flavin reductase (FRed), RebF. We then showed that RebH could be used for preparative halogenation of non-native substrates with catalyst-controlled selectivity. We reported the first examples in which the stability, substrate scope, and site selectivity of a FDH were improved to synthetically useful levels via directed evolution. X-ray crystal structures of evolved FDHs and reversion mutations showed that random mutations throughout the RebH structure were critical to achieving high levels of activity and selectivity on diverse aromatic substrates, and these data were used in combination with molecular dynamics simulations to develop predictive model for FDH selectivity. Finally, we used family wide genome mining to identify a diverse set of FDHs with novel substrate scope and complementary regioselectivity on large, three-dimensionally complex compounds.The diversity of our evolved and mined FDHs allowed us to pursue synthetic applications beyond simple aromatic halogenation. For example, we established that FDHs catalyze enantioselective reactions involving desymmetrization, atroposelective halogenation, and halocyclization. These results highlight the ability of FDH active sites to tolerate different substrate topologies. This utility was further expanded by our recent studies on the single component FDH/FRed, AetF. While we were initially drawn to AetF because it does not require a separate FRed, we found that it halogenates substrates that are not halogenated efficiently or at all by other FDHs and provides high enantioselectivity for reactions that could only be achieved using RebH variants after extensive mutagenesis. Perhaps most notably, AetF catalyzes site-selective aromatic iodination and enantioselective iodoetherification. Together, these studies highlight the origins of FDH engineering, the utility and limitations of the enzymes developed to date, and the promise of FDHs for an ever-expanding range of biocatalytic halogenation reactions.

Structures and Electron Transport Paths in the Four Families of Flavin-Based Electron Bifurcation Enzymes.

Oxidoreductases facilitating electron transfer between molecules are pivotal in metabolic pathways. Flavin-based electron bifurcation (FBEB), a recently discovered energy coupling mechanism in oxidoreductases, enables the reversible division of electron pairs into two acceptors, bridging exergonic and otherwise unfeasible endergonic reactions. This chapter explores the four distinct FBEB complex families and highlights a decade of structural insights into FBEB complexes. In this chapter, we discuss the architecture, electron transfer routes, and conformational changes across all FBEB families, revealing the structural foundation that facilitate these remarkable functions.

Flavin-induced charge separation in transmembrane model peptides.

Hydrophobic peptide models derived from the α-helical transmembrane segment of the epidermal growth factor receptor were synthetically modified with a flavin amino acid as a photo-inducible charge donor and decorated with tryptophans along the helix as charge acceptors. The helical conformation of the peptides was conserved despite the modifications, notably also in lipid vesicles and multibilayers. Their ability to facilitate photo-induced transmembrane charge transport was examined by means of steady-state and time-resolved optical spectroscopy. The first tryptophan next to the flavin donor plays a major role in initiating the charge transport near the N-terminus, while the other tryptophans might promote charge transport along the transmembrane helix. These artificially modified, but still naturally derived helical peptides are important models for studying transmembrane electron transfer and the principles of photosynthesis.

Tuning the Flavin Core via Donor Appendage for Selective Subcellular Bioimaging and PDT Application.

Herein, we report a novel flavin analogue as singular chemical component for lysosome bioimaging, and inherited photosensitizer capability of the flavin core was demonstrated as a promising candidate for photodynamic therapy (PDT) application. Fine-tuning the flavin core with the incorporation of methoxy naphthyl appendage provides an appropriate chemical design, thereby offering photostability, selectivity, and lysosomal colocalization, along with the aggregation-induced emissive nature, making it suitable for lysosomal bioimaging applications. Additionally, photosensitization capability of the flavin core with photostable nature of the synthesized analogue has shown remarkable capacity for generating reactive oxygen species (ROS) within cells, making it a promising candidate for photodynamic therapy (PDT) application.

Surveying the scope of aromatic decarboxylations catalyzed by prenylated-flavin dependent enzymes.

The prenylated-flavin mononucleotide-dependent decarboxylases (also known as UbiD-like enzymes) are the most recently discovered family of decarboxylases. The modified flavin facilitates the decarboxylation of unsaturated carboxylic acids through a novel mechanism involving 1,3-dipolar cyclo-addition chemistry. UbiD-like enzymes have attracted considerable interest for biocatalysis applications due to their ability to catalyse (de)carboxylation reactions on a broad range of aromatic substrates at otherwise unreactive carbon centres. There are now ∼35 000 protein sequences annotated as hypothetical UbiD-like enzymes. Sequence similarity network analyses of the UbiD protein family suggests that there are likely dozens of distinct decarboxylase enzymes represented within this family. Furthermore, many of the enzymes so far characterized can decarboxylate a broad range of substrates. Here we describe a strategy to identify potential substrates of UbiD-like enzymes based on detecting enzyme-catalysed solvent deuterium exchange into potential substrates. Using ferulic acid decarboxylase (FDC) as a model system, we tested a diverse range of aromatic and heterocyclic molecules for their ability to undergo enzyme-catalysed H/D exchange in deuterated buffer. We found that FDC catalyses H/D exchange, albeit at generally very low levels, into a wide range of small, aromatic molecules that have little resemblance to its physiological substrate. In contrast, the sub-set of aromatic carboxylic acids that are substrates for FDC-catalysed decarboxylation is much smaller. We discuss the implications of these findings for screening uncharacterized UbiD-like enzymes for novel (de)carboxylase activity.

Current understanding of enzyme structure and function in bacterial two-component flavin-dependent desulfonases: Cleaving C-S bonds of organosulfur compounds.

The inherent structural properties of enzymes are critical in defining catalytic function. Often, studies to evaluate the relationship between structure and function are limited to only one defined structural element. The two-component flavin-dependent desulfonase family of enzymes involved in bacterial sulfur acquisition utilize a comprehensive range of structural features to carry out the desulfonation of organosulfur compounds. These metabolically essential two-component FMN-dependent desulfonase systems have been proposed to utilize oligomeric changes, protein-protein interactions for flavin transfer, and common mechanistic steps for carbon-sulfur bond cleavage. This review is focused on our current functional and structural understanding of two-component FMN-dependent desulfonase systems from multiple bacterial sources. Mechanistic and structural comparisons from recent independent studies provide fresh insights into the overall functional properties of these systems and note areas in need of further investigation. The review acknowledges current studies focused on evaluating the structural properties of these enzymes in relationship to their distinct catalytic function. The role of these enzymes in maintaining adequate sulfur levels, coupled with the conserved nature of these enzymes in diverse bacteria, underscore the importance in understanding the functional and structural nuances of these systems.

Mechanism of Nitrone Formation by a Flavin-Dependent Monooxygenase.

OxaD is a flavin-dependent monooxygenase (FMO) responsible for catalyzing the oxidation of an indole nitrogen atom, resulting in the formation of a nitrone. Nitrones serve as versatile intermediates in complex syntheses, including challenging reactions like cycloadditions. Traditional organic synthesis methods often yield limited results and involve environmentally harmful chemicals. Therefore, the enzymatic synthesis of nitrone-containing compounds holds promise for more sustainable industrial processes. In this study, we explored the catalytic mechanism of OxaD using a combination of steady-state and rapid-reaction kinetics, site-directed mutagenesis, spectroscopy, and structural modeling. Our investigations showed that OxaD catalyzes two oxidations of the indole nitrogen of roquefortine C, ultimately yielding roquefortine L. The reductive-half reaction analysis indicated that OxaD rapidly undergoes reduction and follows a "cautious" flavin reduction mechanism by requiring substrate binding before reduction can take place. This characteristic places OxaD in class A of the FMO family, a classification supported by a structural model featuring a single Rossmann nucleotide binding domain and a glutathione reductase fold. Furthermore, our spectroscopic analysis unveiled both enzyme-substrate and enzyme-intermediate complexes. Our analysis of the oxidative-half reaction suggests that the flavin dehydration step is the slow step in the catalytic cycle. Finally, through mutagenesis of the conserved D63 residue, we demonstrated its role in flavin motion and product oxygenation. Based on our findings, we propose a catalytic mechanism for OxaD and provide insights into the active site architecture within class A FMOs.

Deazaflavin metabolite produced by endosymbiotic bacteria controls fungal host reproduction.

The endosymbiosis between the pathogenic fungus Rhizopus microsporus and the toxin-producing bacterium Mycetohabitans rhizoxinica represents a unique example of host control by an endosymbiont. Fungal sporulation strictly depends on the presence of endosymbionts as well as bacterially produced secondary metabolites. However, an influence of primary metabolites on host control remained unexplored. Recently, we discovered that M. rhizoxinica produces FO and 3PG-F420, a derivative of the specialized redox cofactor F420. Whether FO/3PG-F420 plays a role in the symbiosis has yet to be investigated. Here, we report that FO, the precursor of 3PG-F420, is essential to the establishment of a stable symbiosis. Bioinformatic analysis revealed that the genetic inventory to produce cofactor 3PG-F420 is conserved in the genomes of eight endofungal Mycetohabitans strains. By developing a CRISPR/Cas-assisted base editing strategy for M. rhizoxinica, we generated mutant strains deficient in 3PG-F420 (M. rhizoxinica ΔcofC) and in both FO and 3PG-F420 (M. rhizoxinica ΔfbiC). Co-culture experiments demonstrated that the sporulating phenotype of apo-symbiotic R. microsporus is maintained upon reinfection with wild-type M. rhizoxinica or M. rhizoxinica ΔcofC. In contrast, R. microsporus is unable to sporulate when co-cultivated with M. rhizoxinica ΔfbiC, even though the fungus was observed by super-resolution fluorescence microscopy to be successfully colonized. Genetic and chemical complementation of the FO deficiency of M. rhizoxinica ΔfbiC led to restoration of fungal sporulation, signifying that FO is indispensable for establishing a functional symbiosis. Even though FO is known for its light-harvesting properties, our data illustrate an important role of FO in inter-kingdom communication.

Oxygen-transfer reactions by enzymatic flavin-N5 oxygen adducts-Oxidation is not a must.

Flavoenzymes catalyze numerous redox reactions including the transfer of an O2-derived oxygen atom to organic substrates, while the other one is reduced to water. Investigation of some of these monooxygenases led to a detailed understanding of their catalytic cycle, which involves the flavin-C4α-(hydro)peroxide as hallmark oxygenating species, and newly discovered flavoprotein monooxygenases were generally assumed to operate similarly. However, discoveries in recent years revealed a broader mechanistic versatility, including enzymes that utilize flavin-N5 oxygen adducts for catalysis in the form of the flavin-N5-(hydro)peroxide and the flavin-N5-oxide species. In this review, I will highlight recent developments in that area, including noncanonical flavoenzymes from natural product biosynthesis and sulfur metabolism that provide first insights into the chemical properties of these species. Remarkably, some enzymes may even combine the flavin-N5-peroxide and the flavin-N5-oxide species for consecutive oxygen-transfers to the same substrate and thereby in essence operate as dioxygenases.

The Promiscuous Flavin-Dependent Monooxygenase PboD from Aspergillus ustus Increases the Structural Diversity of Hydroxylated Pyrroloindoline Diketopiperazines.

The potential of natural products as pharmaceutical and agricultural agents is based on their large structural diversity, resulting in part from modifications of the backbone structure by tailoring enzymes during biosynthesis. Flavin-dependent monooxygenases (FMOs), as one such group of enzymes, play an important role in the biosynthesis of diverse natural products, including cyclodipeptide (CDP) derivatives. The FMO PboD was shown to catalyze C-3 hydroxylation at the indole ring of cyclo-l-Trp-l-Leu in the biosynthesis of protubonines, accompanied by pyrrolidine ring formation. PboD substrate promiscuity was investigated in this study by testing its catalytic activity toward additional tryptophan-containing CDPs in vitro and biotransformation in Aspergillus nidulans transformants bearing a truncated protubonine gene cluster with pboD and two acetyltransferase genes. High acceptance of five CDPs was detected for PboD, especially of those with a second aromatic moiety. Isolation and structure elucidation of five pyrrolidine diketopiperazine products, with two new structures, proved the expected stereospecific hydroxylation and pyrrolidine ring formation. Determination of kinetic parameters revealed higher catalytic efficiency of PboD toward three CDPs consisting of aromatic amino acids than of its natural substrate cyclo-l-Trp-l-Leu. In the biotransformation experiments with the A. nidulans transformant, modest formation of hydroxylated and acetylated products was also detected.

X-ray structure and enzymatic study of a bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX.

NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2's requirement for activation.

Tri-enzyme fusion of tryptophan halogenase achieves a concise strategy for coenzyme self-sufficiency and the continuous halogenation of L-tryptophan.

The halogenase-based catalysis is one of the most environmentally friendly methods for the synthesis of halogenated products, among which flavin-dependent halogenases (FDHs) have attracted great interest as one of the most promising biocatalysts due to the remarkable site-selectivity and wide substrate range. However, the complexity of constructing the NAD+-NADH-FAD-FADH2 bicoenzyme cycle system has affected the engineering applications of FDHs. In this work, a coenzyme self-sufficient tri-enzyme fusion was constructed and successfully applied to the continuous halogenation of L-tryptophan. SpFDH was firstly identified derived from Streptomyces pratensis, a highly selective halogenase capable of generating 6-chloro-tryptophan from tryptophan. Then, using gene fusion technology, SpFDH was fused with glucose dehydrogenase (GDH) and flavin reductase (FR) to form a tri-enzyme fusion, which increased the yield by 1.46-fold and making the coenzymes self-sufficient. For more efficient halogenation of L-tryptophan, a continuous halogenation bioprocess of L-tryptophan was developed by immobilizing the tri-enzyme fusion and attaching it to a continuous catalytic device, which resulted in a reaction yield of 97.6% after 12 h reaction. An FDH from S. pratensis was successfully applied in the halogenation and our study provides a concise strategy for the preparation of halogenated tryptophan mediated by multienzyme cascade catalysis.

Systematic Analysis of the Effect of Genomic Knock-Out of Non-Essential Promiscuous HAD-Like Phosphatases YcsE, YitU and YwtE on Flavin and Adenylate Content in Bacillus Subtilis.

Studying the metabolic role of non-essential promiscuous enzymes is a challenging task, as genetic manipulations usually do not reveal at which point(s) of the metabolic network the enzymatic activity of such protein is beneficial for the organism. Each of the HAD-like phosphatases YcsE, YitU and YwtE of Bacillus subtilis catalyzes the dephosphorylation of 5-amino-6-ribitylamino-uracil 5'-phosphate, which is essential in the biosynthesis of riboflavin. Using CRISPR technology, we have found that the deletion of these genes, individually or in all possible combinations failed to cause riboflavin auxotrophy and did not result in significant growth changes. Analysis of flavin and adenylate content in B. subtilis knockout mutants showed that (i) there must be one or several still unidentified phosphatases that can replace the deleted proteins; (ii) such replacements, however, cannot fully restore the intracellular content of any of three flavins studied (riboflavin, FMN, FAD); (iii) whereas bacterial fitness was not significantly compromised by mutations, the intracellular balance of flavins and adenylates did show some significant changes.