A flavin-dependent tryptophan 6-halogenase and its use in modification of pyrrolnitrin biosynthesis

Regioselective halogenation of electron rich substrates is catalysed by flavin-dependent halogenases. Thienodolin produced by Streptomyces albogriseolus contains a chlorine atom in the 6-position of the indole ring system and is believed to be derived from tryptophan. Using the gene of the tryptophan 7-halogenase (PrnA) from the pyrrolnitrin biosynthetic gene cluster the gene for a tryptophan 6-halogenase was cloned, sequenced and heterologously overexpressed in Pseudomonas strains. In vitro activity of the purified enzyme could only be shown in a two-component enzyme system consisting of the halogenase, a flavin reductase, NADH, FAD and halide ions. The enzyme catalyses the regioselective chlorination and bromination of l- and d-tryptophan. In its native form the enzyme is probably a homodimer with a relative molecular mass of the subunits of 63 600 (including the His-tag). Transformation of the pyrrolnitrin producer Pseudomonas chlororaphis ACN with a plasmid containing the tryptophan 6-halogenase gene lead to the formation of the new aminopyrrolnitrin derivative 3-(2′-amino-4′-chlorophenyl) pyrrole.     

Structure and Action of the Myxobacterial Chondrochloren Halogenase CndH: A New Variant of FAD-dependent Halogenases

The crystal structure of the FAD-dependent chondrochloren halogenase CndH has been established at 2.1 Å resolution. The enzyme contains the characteristic FAD-binding scaffold of the glutathione reductase superfamily. Except for its C-terminal domain, the chainfold of CndH is virtually identical with those of FAD-dependent aromatic hydroxylases. When compared to the structurally known FAD-dependent halogenases PrnA and RebH, CndH lacks a 45 residue segment near position 100 and deviates in the C-terminal domain. Both variations are near the active center and appear to reflect substrate differences. Whereas PrnA and RebH modify free tryptophan, CndH halogenates the tyrosyl group of a chondrochloren precursor that is most likely bound to a carrier protein. In contrast to PrnA and RebH, which enclose their small substrate completely, CndH has a large non-polar surface patch that may accommodate the putative carrier. Apart from the substrate binding site, the active center of CndH corresponds to those of PrnA and RebH. At the halogenation site, CndH has the characteristic lysine (Lys76) but lacks the required base Glu346 (PrnA). This base may be supplied by a residue of its C-terminal domain or by the carrier. These differences were corroborated by an overall sequence comparison between the known FAD-dependent halogenases, which revealed a split into a PrnA-RebH group and a CndH group. The two functionally established members of the CndH group use carrier-bound substrates, whereas three members of PrnA-RebH group are known to accept a free amino acid. Given the structural and functional distinction, we classify CndH as a new variant B of the FAD-dependent halogenases, adding a new feature to the structurally established variant A enzymes PrnA and RebH.     

Binding of FAD and tryptophan to the tryptophan 6‐halogenase Thal is negatively coupled

Flavin‐dependent halogenases require reduced flavin adenine dinucleotide (FADH₂), O₂, and halide salts to halogenate their substrates. We describe the crystal structures of the tryptophan 6‐halogenase Thal in complex with FAD or with both tryptophan and FAD. If tryptophan and FAD were soaked simultaneously, both ligands showed impaired binding and in some cases only the adenosine monophosphate or the adenosine moiety of FAD was resolved, suggesting that tryptophan binding increases the mobility mainly of the flavin mononucleotide moiety. This confirms a negative cooperativity between the binding of substrate and cofactor that was previously described for other tryptophan halogenases. Binding of substrate to tryptophan halogenases reduces the affinity for the oxidized cofactor FAD presumably to facilitate the regeneration of FADH₂ by flavin reductases.     

Chloramphenicol Biosynthesis: The Structure of CmlS, a Flavin-Dependent Halogenase Showing a Covalent Flavin-Aspartate Bond

Chloramphenicol is a halogenated natural product bearing an unusual dichloroacetyl moiety that is critical for its antibiotic activity. The operon for chloramphenicol biosynthesis in Streptomyces venezuelae encodes the chloramphenicol halogenase CmlS, which belongs to the large and diverse family of flavin-dependent halogenases (FDH’s). CmlS was previously shown to be essential for the formation of the dichloroacetyl group. Here we report the X-ray crystal structure of CmlS determined at 2.2 Å resolution, revealing a flavin monooxygenase domain shared by all FDHs, but also a unique ‘winged-helix’ C-terminal domain that creates a T-shaped tunnel leading to the halogenation active site. Intriguingly, the C-terminal tail of this domain blocks access to the halogenation active site, suggesting a structurally dynamic role during catalysis. The halogenation active site is notably nonpolar and shares nearly identical residues with Chondromyces crocatus tyrosyl halogenase (CndH), including the conserved Lys (K71) that forms the reactive chloramine intermediate. The exception is Y350, which could be used to stabilize enolate formation during substrate halogenation. The strictly conserved residue E44, located near the isoalloxazine ring of the bound flavin adenine dinucleotide (FAD) cofactor, is optimally positioned to function as a remote general acid, through a water-mediated proton relay, which could accelerate the reaction of the chloramine intermediate during substrate halogenation, or the oxidation of chloride by the FAD(C4α)-OOH intermediate. Strikingly, the 8α carbon of the FAD cofactor is observed to be covalently attached to D277 of CmlS, a residue that is highly conserved in the FDH family. In addition to representing a new type of flavin modification, this has intriguing implications for the mechanism of FDHs. Based on the crystal structure and in analogy to known halogenases, we propose a reaction mechanism for CmlS.     

Flavin-dependent halogenases involved in secondary metabolism in bacteria

The understanding of biological halogenation has increased during the last few years. While haloperoxidases were the only halogenating enzymes known until 1997, it is now clear that haloperoxidases are hardly, if at all, involved in biosynthesis of more complex halogenated compounds in microorganisms. A novel type of halogenating enzymes, flavin-dependent halogenases, has been identified as a major player in the introduction of chloride and bromide into activated organic molecules. Flavin-dependent halogenases require the activity of a flavin reductase for the production of reduced flavin, required by the actual halogenase. A number of flavin-dependent tryptophan halogenases have been investigated in some detail, and the first three-dimensional structure of a member of this enzyme subfamily, tryptophan 7-halogenase, has been elucidated. This structure suggests a mechanism involving the formation of hypohalous acid, which is used inside the enzyme for regioselective halogenation of the respective substrate. The introduction of halogen atoms into non-activated alkyl groups is catalysed by non-heme Fe^II α-ketoglutarate- and O₂-dependent halogenases. Examples for the use of flavin-dependent halogenases for the formation of novel halogenated compounds in in vitro and in vivo reactions promise a bright future for the application of biological halogenation reactions.     

Chlorination by a long-lived intermediate in the mechanism of flavin-dependent halogenases

The flavin-dependent halogenase RebH catalyzes the formation of 7-chlorotryptophan as the initial step in the biosynthesis of antitumor agent rebeccamycin. The reaction of FADH2, Cl-, and O2 in the active site generates the powerful oxidant HOCl, which was presumed to carry out the chlorination reaction. Herein, we demonstrate the formation of a long-lived chlorinating intermediate (t1/2 = 63 h at 4 degrees C) when RebH, FADH2, Cl-, and O2 react in the absence of substrate tryptophan. This intermediate remained on the enzyme after removal of FAD and transferred chlorine to tryptophan with kinetically competent rates. The identity of this intermediate is suggested by the X-ray crystal structure of RebH, which revealed an active site Lys79 located in a central position between flavin and tryptophan binding sites and just 4.1 A above C7 of tryptophan. The chlorinating species is proposed to be a Lys-epsilonNH-Cl (lysine chloramine) from reaction of enzyme-generated HOCl with the active site Lys79. This covalent enzyme chloramine likely plays a key role in directing regiospecific chlorination of substrate in this important class of biosynthetic enzymes.     

Adenosine diphosphate moiety does not participate in structural changes for the signaling state in the sensor of blue-light using FAD domain of AppA

A sensor of blue light using FAD (BLUF) protein is a flavin adenine dinucleotide (FAD) based new class blue-light sensory flavoprotein. The BLUF domain of AppA was reconstituted in vitro from apoprotein and flavin adenine dinucleotide, flavin adenine mononucleotide or riboflavin. The light-induced FTIR spectra of the domain reconstituted from various flavins and the 13C-labeled apoprotein showed that identical light-induced structural changes occur in both the flavin chromophore and protein for the signaling state in all of the reconstituted holoproteins. The results showed that an adenosine 5'-dinucleotide moiety is not required for signaling-state formation in a BLUF domain.     

Elucidations of the Catalytic Cycle of NADH-Cytochrome b5 Reductase by X-ray Crystallography: New Insights into Regulation of Efficient Electron Transfer

NADH-Cytochrome b₅ reductase (b5R), a flavoprotein consisting of NADH and flavin adenine dinucleotide (FAD) binding domains, catalyzes electron transfer from the two-electron carrier NADH to the one-electron carrier cytochrome b₅ (Cb5). The crystal structures of both the fully reduced form and the oxidized form of porcine liver b5R were determined. In the reduced b5R structure determined at 1.68 Å resolution, the relative configuration of the two domains was slightly shifted in comparison with that of the oxidized form. This shift resulted in an increase in the solvent-accessible surface area of FAD and created a new hydrogen-bonding interaction between the N5 atom of the isoalloxazine ring of FAD and the hydroxyl oxygen atom of Thr66, which is considered to be a key residue in the release of a proton from the N5 atom. The isoalloxazine ring of FAD in the reduced form is flat as in the oxidized form and stacked together with the nicotinamide ring of NAD⁺. Determination of the oxidized b5R structure, including the hydrogen atoms, determined at 0.78 Å resolution revealed the details of a hydrogen-bonding network from the N5 atom of FAD to His49 via Thr66. Both of the reduced and oxidized b5R structures explain how backflow in this catalytic cycle is prevented and the transfer of electrons to one-electron acceptors such as Cb5 is accelerated. Furthermore, crystallographic analysis by the cryo-trapping method suggests that re-oxidation follows a two-step mechanism. These results provide structural insights into the catalytic cycle of b5R.     

Sequence-structure analysis of FAD-containing proteins

We have analyzed structure-sequence relationships in 32 families of flavin adenine dinucleotide (FAD)-binding proteins, to prepare for genomic-scale analyses of this family. Four different FAD-family folds were identified, each containing at least two or more protein families. Three of these families, exemplified by glutathione reductase (GR), ferredoxin reductase (FR), and p-cresol methylhydroxylase (PCMH) were previously defined, and a family represented by pyruvate oxidase (PO) is newly defined. For each of the families, several conserved sequence motifs have been characterized. Several newly recognized sequence motifs are reported here for the PO, GR, and PCMH families. Each FAD fold can be uniquely identified by the presence of distinctive conserved sequence motifs. We also analyzed cofactor properties, some of which are conserved within a family fold while others display variability. Among the conserved properties is cofactor directionality: in some FAD-structural families, the adenine ring of the FAD points toward the FAD-binding domain, whereas in others the isoalloxazine ring points toward this domain. In contrast, the FAD conformation and orientation are conserved in some families while in others it displays some variability. Nevertheless, there are clear correlations among the FAD-family fold, the shape of the pocket, and the FAD conformation. Our general findings are as follows: (a) no single protein 'pharmacophore' exists for binding FAD; (b) in every FAD-binding family, the pyrophosphate moiety binds to the most strongly conserved sequence motif, suggesting that pyrophosphate binding is a significant component of molecular recognition; and (c) sequence motifs can identify proteins that bind phosphate-containing ligands.     

Flavin redox chemistry precedes substrate chlorination during the reaction of the flavin-dependent halogenase RebH

The flavin-dependent halogenase RebH catalyzes chlorination at the C7 position of tryptophan as the initial step in the biosynthesis of the chemotherapeutic agent rebeccamycin. The reaction requires reduced FADH(2) (provided by a partner flavin reductase), chloride ion, and oxygen as cosubstrates. Given the similarity of its sequence to those of flavoprotein monooxygenases and their common cosubstrate requirements, the reaction of FADH(2) and O(2) in the halogenase active site was presumed to form the typical FAD(C4a)-OOH intermediate observed in monooxygenase reactions. By using stopped-flow spectroscopy, formation of a FAD(C4a)-OOH intermediate was detected during the RebH reaction. This intermediate decayed to yield a FAD(C4a)-OH intermediate. The order of addition of FADH(2) and O(2) was critical for accumulation of the FAD(C4a)-OOH intermediate and for subsequent product formation, indicating that conformational dynamics may be important for protection of labile intermediates formed during the reaction. Formation of flavin intermediates did not require tryptophan, nor were their rates of formation affected by the presence of tryptophan, suggesting that tryptophan likely does not react directly with any flavin intermediates. Furthermore, although final oxidation to FAD occurred with a rate constant of 0.12 s(-)(1), quenched-flow kinetic data showed that the rate constant for 7-chlorotryptophan formation was 0.05 s(-)(1) at 25 degrees C. The kinetic analysis establishes that substrate chlorination occurs after completion of flavin redox reactions. These findings are consistent with a mechanism whereby hypochlorite is generated in the RebH active site from the reaction of FADH(2), chloride ion, and O(2).     

The succinate dehydrogenase assembly factor,SdhE, is required for the flavinylation and activation of fumarate reductase in bacteria

The activity of the respiratory enzyme fumarate reductase (FRD) is dependent on the covalent attachment of the redox cofactor flavin adenine dinucleotide (FAD). We demonstrate that the FAD assembly factor SdhE, which flavinylates and activates the respiratory enzyme succinate dehydrogenase (SDH), is also required for the complete activation and flavinylation of FRD. SdhE interacted with, and flavinylated, the flavoprotein subunit FrdA, whilst mutations in a conserved RGxxE motif impaired the complete flavinylation and activation of FRD. These results are of widespread relevance because SDH and FRD play an important role in cellular energetics and are required for virulence in many important bacterial pathogens.     

Catalytic mechanisms, basic roles, and biotechnological and environmental significance of halogenating enzymes

The understanding of enzymatic incorporation of halogen atoms into organic molecules has increased during the last few years. Two novel types of halogenating enzymes, flavindependent halogenases and α-ketoglutarate-dependent halogenases, are now known to play a significant role in enzyme-catalyzed halogenation. The recent advances on the halogenating enzymes RebH, SyrB2, and CytC3 have suggested some new mechanisms for enzymatic halogenations. This review concentrates on the occurrence, catalytic mechanisms, and biotechnological applications of the halogenating enzymes that are currently known.     

A Fivefold Parallelized Biosynthetic Process Secures Chlorination of Armillaria mellea (Honey Mushroom) Toxins

The basidiomycetous tree pathogen Armillaria mellea (honey mushroom) produces a large variety of structurally related antibiotically active and phytotoxic natural products, referred to as the melleolides. During their biosynthesis, some members of the melleolide family of compounds undergo monochlorination of the aromatic moiety, whose biochemical and genetic basis was not known previously. This first study on basidiomycete halogenases presents the biochemical in vitro characterization of five flavin-dependent A. mellea enzymes (ArmH1 to ArmH5) that were heterologously produced in Escherichia coli. We demonstrate that all five enzymes transfer a single chlorine atom to the melleolide backbone. A 5-fold, secured biosynthetic step during natural product assembly is unprecedented. Typically, flavin-dependent halogenases are categorized into enzymes acting on free compounds as opposed to those requiring a carrier-protein-bound acceptor substrate. The enzymes characterized in this study clearly turned over free substrates. Phylogenetic clades of halogenases suggest that all fungal enzymes share an ancestor and reflect a clear divergence between ascomycetes and basidiomycetes.     

Tryptophan 7-Halogenase from Pseudomonas aureofaciens ACN Strain: Gene Cloning and Sequencing and the Enzyme Expression

The gene of tryptophan 7-halogenase was isolated from the Pseudomonas aureofaciens ACN strain producing pyrrolnitrin, a chlorocontaining antibiotic, and sequenced. A high homology degree (over 95%) was established for the genes and the corresponding halogenases from P. aureofaciens ACN and P. fluorescens BL915. The tryptophan 7-halogenase gene was amplified by PCR, and the corresponding enzyme was expressed in Escherichia coli cells using the pBSII SK+ vector.     

Regulation of flavin biosynthesis in the methylotrophic yeast Hansenula polymorpha

Under various conditions of growth of the methylotrophic yeast Hansenula polymorpha, a tight correlation was observed between the levels of flavin adenine dinucleotide (FAD)-containing alcohol oxidase, and the levels of intracellularly bound FAD and flavin biosynthetic enzymes. Adaptation of the organism to changes in the physiological requirement for FAD was by adjustment of the levels of the enzymes catalyzing the last three steps in flavin biosynthesis, riboflavin synthetase, riboflavin kinase and flavin mononucleotide adenylyltransferase. The regulation of the synthesis of the latter enzymes in relation to that of alcohol oxidase synthesis was studied in experiments involving addition of glucose to cells of H. polymorpha growing on methanol in batch cultures or in carbon-limited continuous cultures. This resulted not only in selective inactivation of alcohol oxidase and release of FAD, as previously reported, but invariably also in repression/inactivation of the flavin biosynthetic enzymes. In further experiments involving addition of FAD to the same type of cultures it became clear that inactivation of the latter enzymes was not caused directly by glucose, but rather by free FAD that accumulated intracellularly. In these experiments no repression or inactivation of alcohol oxidase occurred and it is therefore concluded that the synthesis of this enzyme and the flavin biosynthetic enzymes is under separate control, the former by glucose (and possibly methanol) and the latter by intracellular levels of free FAD.Abbreviations FAD Flavin adenine dinucleotide - FMN riboflavin-5-phosphate; flavin mononucleotide - Rf riboflavin     

Mechanism-based inhibitors of cytokinin oxidase/dehydrogenase attack FAD cofactor

Cytokinin oxidases/dehydrogenases (CKOs) mediate catabolic regulation of cytokinin levels in plants. Several substrate analogs containing an unsaturated side chain were studied for their possible inhibitory effect on maize CKO (ZmCKO1) by use of various bioanalytical methods. Two allenic derivatives, N⁶-(buta-2,3-dienyl)adenine (HA-8) and N⁶-(penta-2,3-dienyl)adenine (HA-1), were identified as strong mechanism-based inhibitors of the enzyme. Despite exhaustive dialysis, the enzyme remained inhibited. Conversely, substrate analogs with a triple bond in the side chain were much weaker inactivators. The crystal structures of recombinant ZmCKO1 complexed with HA-1 or HA-8 were solved to 1.95 Å resolution. Together with Raman spectra of the inactivated enzyme, it was revealed that reactive imine intermediates generated by oxidation of the allenic inhibitors covalently bind to the flavin adenine dinucleotide (FAD) cofactor. The binding occurs at the C4a atom of the isoalloxazine ring of FAD, the planarity of which is consequently disrupted. All the compounds under study were also analyzed for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4 in a bacterial receptor assay and for cytokinin activity in the Amaranthus bioassay. HA-1 and HA-8 were found to be good receptor ligands with a significant cytokinin activity. Nevertheless, due to their ability to inactivate CKO in the desired time intervals or developmental stages, they both represent attractive compounds for physiological studies, as the inhibition mechanism of HA-1 and HA-8 is mainly FAD dependent.     

Structural Features of Human Monoamine Oxidase A Elucidated from cDNA and Peptide Sequences

Monoamine oxidase (MAO), an important enzyme for the degradation of amine neurotransmitters, has been implicated in neuropsychiatric illness. The amino acid sequence for one form of the enzyme, MAO-A, has been deduced from human cDNA clones and verified against proteolytic peptides. The covalent binding site for the flavin adenine dinucleotide (FAD) cofactor is near the C-terminal region. The presence of features characteristic of the ADP-binding fold suggests that the N-terminal region is also involved in the binding of FAD. These cDNAs should facilitate the study of the structure, function, and intracellular targeting of MAO, as well as the analysis of its expression in normal and pathological states.     

Oligomeric State in the Crystal Structure of Modular FAD Synthetase Provides Insights into Its Sequential Catalysis in Prokaryotes

The crystal structure of the modular flavin adenine dinucleotide (FAD) synthetase from Corynebacterium ammoniagenes has been solved at 1.95 Å resolution. The structure of C. ammoniagenes FAD synthetase presents two catalytic modules—a C-terminus with ATP-riboflavin kinase activity and an N-terminus with ATP-flavin mononucleotide (FMN) adenylyltransferase activity—that are responsible for the synthesis of FAD from riboflavin in two sequential steps. In the monomeric structure, the active sites from both modules are placed 40 Å away, preventing the direct transfer of the product from the first reaction (FMN) to the second catalytic site, where it acts as substrate. Crystallographic and biophysical studies revealed a hexameric assembly formed by the interaction of two trimers. Each trimer presents a head-tail configuration, with FMN adenylyltransferase and riboflavin kinase modules from different protomers approaching the active sites and allowing the direct transfer of FMN. Experimental results provide molecular-level evidences of the mechanism of the synthesis of FMN and FAD in prokaryotes in which the oligomeric state could be involved in the regulation of the catalytic efficiency of the modular enzyme.     

黄素依赖型卤化酶的结构特点及工程改造*

卤化物是通过卤化酶催化卤族元素在有机化合物上特定位置发生取代形成的一类化合物,具有独特的生理生化作用。黄素依赖型卤化酶具有良好的区域选择性,虽然有相似的黄素分子的结合位点,但在底物结合方面略有不同,对其结构和合成途径及结合蛋白质工程的随机诱变和定向改造的研究在工业应用中至关重要。讨论了具有高区域选择性的黄素依赖型卤化酶的结构特点及工程改造,以及经过工程改造后黄素依赖型卤化酶在工业生产中的应用。     

Gain of function in an ERV/ALR sulfhydryl oxidase by molecular engineering of the shuttle disulfide

The ERV/ALR sulfhydryl oxidase domain is a versatile module adapted for catalysis of disulfide bond formation in various organelles and biological settings. Its four-helix bundle structure juxtaposes a Cys-X-X-Cys dithiol/disulfide motif with a bound flavin adenine dinucleotide (FAD) cofactor, enabling transfer of electrons from thiol substrates to non-thiol electron acceptors. ERV/ALR family members contain an additional di-cysteine motif outside the four-helix-bundle core. Although the location and context of this "shuttle" disulfide differs among family members, it is proposed to perform the same basic function of mediating electron transfer from substrate to the enzyme active site. We have determined by X-ray crystallography the structure of AtErv1, an ERV/ALR enzyme that contains a Cys-X4-Cys shuttle disulfide and oxidizes thioredoxin in vitro, and compared it to ScErv2, which has a Cys-X-Cys shuttle and does not oxidize thioredoxin at an appreciable rate. The AtErv1 shuttle disulfide is in a region of the structure that is disordered and thus apparently mobile and exposed. This feature may facilitate access of protein substrates to the shuttle disulfide. To test whether the shuttle disulfide region is modular and can confer on other enzymes oxidase activity toward new substrates, we generated chimeric enzyme variants combining shuttle disulfide and core elements from AtErv1 and ScErv2 and monitored oxidation of thioredoxin by the chimeras. We found that the AtErv1 shuttle disulfide region could indeed confer thioredoxin oxidase activity on the ScErv2 core. Remarkably, various chimeras containing the ScErv2 Cys-X-Cys shuttle disulfide were found to function efficiently as well. Since neither the ScErv2 core nor the Cys-X-Cys motif is therefore incapable of participating in oxidation of thioredoxin, we conclude that wild-type ScErv2 has evolved to repress activity on substrates of this type, perhaps in favor of a different, as yet unknown, substrate.