Vanadium-dependent Haloperoxidases Research Articles

Synthesis, structure, and catalytic bromination of supramolecular oxovanadium complexes containing oxalate

Three supramolecular complexes, [VO(phen)(C2O4)(H2O)]·CH3OH (1) [(VO)2(u2-C2O4)(C2O4)2(H2O)2]·L·H2O (2), and [(4,4′-bipyH2)0.5]+[VO2(2,6-dipic)]−·2H2O (3) (phen = 1,10-phenanthroline 4,4′-bipy = 4,4′-bipyridine, 2,6-dipic = 2,6-pyridinedicarboxylic, L = 1,4-bis((3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzene), have been prepared and characterized by elemental analysis, IR, and UV–vis spectroscopy and single-crystal diffraction analysis. Structural analysis shows that the three complexes all contain carboxylate and V=O moiety; vanadium of 1 and 2 are six coordinate with distorted octahedral geometry with N2O4 and O6 donor sets, respectively, while 3 is five coordinate with distorted trigonal bipyramidal geometry with a NO4 donor set. The complexes exhibit catalytic bromination activity in the single-pot reaction for the conversion of phenol red to bromophenol blue in H2O–DMF at 30 ± 0.5 C with pH 5.8, indicating that they can be considered as functional model vanadium-dependent haloperoxidases. In addition, electrochemical behaviors are also studied.

The future of/for vanadium

Vanadium compounds are stored or employed by several groups of bacterial and eukaryotic organisms. Two types of vanadium-dependent enzymes have so far been characterised: vanadate-dependent haloperoxidases from fungi, lichens, marine macroalgae and Streptomyces bacteria, and vanadium nitrogenases in proteo- and cyanobacteria. Several bacterial strains can employ vanadate(V) as an external electron acceptor in respiration, reducing vanadate to VO(2+) and thus contributing to the mineralisation of vanadium and to the detoxification of vanadate-contaminated water. Amanita mushrooms and many sea squirts accumulate vanadium, without the importance of this practise being well understood. Further, the analogy between vanadate and phosphate implicates an interference of vanadate with metabolic processes involving phosphate, suggesting a regulatory role for vanadate in most if not all organisms, including humans, but also hinting at toxic effects at unphysiologically high vanadate concentrations. The antidiabetic effect of vanadium compounds is probably related to the phosphate-vanadate antagonism, as is the potentiality of vanadate in the amelioration of cardiovascular affliction. The anti-cancer action of vanadium compounds and their in vitro activity towards the protozoa causing amoebiasis, leishmaniasis and Chagas' disease again may be rooted in the intervention of vanadate with the activity of phosphatases and kinases. In addition, most likely the ability of vanadate(V) and oxidovanadium(IV) to regulate the cellular production of reactive oxygen species comes in, thus influencing cellular signalling. Future developments of vanadium chemistry are likely to emphasize topics related to biological, environmental and medicinal aspects. Condensation of monovanadate results in the formation of oligovanadates, polyvanadates and finally colloidal and solid vanadium oxides that, in part, convey bio-mimetic functions comparable to those of simple vanadate, including its catalytic potential as an active centre in haloperoxidases and the lethal action against viruses, bacteria and protozoan parasites. Decavanadate has been shown to be stabilised by docking to proteins, and by integration into nanoscopic water pools of intracellular compartments, modelled by reverse micelles. The well established and approved use of vanadium oxides in, amongst other applications, catalysis has been recently impacted by the elucidation of the active surface species--VO(x)--of catalysts based on mixed vanadium oxides, and vanadium oxides on supports. Finally, materials based on vanadium oxides and vanadates play an increasingly important role as cathode materials in high density lithium batteries. An example is Ag2VO2PO4, which, in the discharge process, is reduced to Li(3.2)VO2PO4 and Ag. Oncoming developments in vanadium chemistry thus include oxide-based materials.

Vanadium complexes having [VO]2+, [VO]3+ and [VO2]+ cores with hydrazones of 2,6-diformyl-4-methylphenol: synthesis, characterization, reactivity, and catalytic potential

The Schiff bases H3dfmp(L)2 obtained by the condensation of 2,6-diformyl-4-methylphenol and hydrazones [L = isonicotinoylhydrazide (inh), nicotinoylhydrazide (nah) and benzoylhydrazide (bhz)] are prepared and characterized. By reaction of [V(IV)O(acac)2] and the H3dfmp(L)2 in methanol the V(IV)O-complexes [V(IV)O{Hdfmp(inh)2}(H2O)] (1), [V(IV)O{Hdfmp(nah)2}(H2O)] (2) and [V(IV)O{Hdfmp(bhz)2}(H2O)] (3) were obtained. Upon their aerial oxidation in methanol [V(V)O(OMe)(MeOH){Hdfmp(inh)2}] (4), [V(V)O(OMe)(MeOH){Hdfmp(nah)2}] (5) and [V(V)O(OMe)(MeOH){Hdfmp(bhz)2}] (6) were isolated. In the presence of KOH, oxidation of 1-3 results in the formation of [V(V)O2{H2dfmp(inh)2}]n·5H2O (7), K[V(V)O2{Hdfmp(nah)2}] (8) and K[V(V)O2{Hdfmp(bhz)2}] (9). All compounds are characterized in the solid state and in solution, namely by spectroscopic techniques (IR, UV-Vis, EPR, (1)H, (13)C and (51)V NMR), and DFT is also used to calculate the V(IV) hyperfine coupling constants of V(IV)-compounds and (51)V NMR chemical shifts of several V(V)-species and assign them to those formed in solution. Single crystal X-ray analysis of [V(V)O(OMe)(MeOH){Hdfmp(bhz)2}] (6) and [V(V)O2{H2dfmp(inh)2}]n·5H2O (7) confirm the coordination of the ligand in the dianionic (ONO(2-)) enolate tautomeric form, one of the hydrazide moieties remaining non-coordinated. In the case of 7 the free N(pyridine) atom of the inh moiety coordinates to the other vanadium center yielding a polynuclear complex in the solid state. It is also demonstrated that the V(V)O2-complexes are catalyst precursors in the oxidative bromination of styrene by H2O2, therefore acting as functional models of vanadium dependent haloperoxidases. Plausible intermediates involved in the catalytic process are established by UV-Vis, (51)V NMR and DFT studies.

Merochlorins A–D, Cyclic Meroterpenoid Antibiotics Biosynthesized in Divergent Pathways with Vanadium-Dependent Chloroperoxidases

Meroterpenoids are mixed polyketide-terpenoid natural products with a broad range of biological activities. Herein, we present the structures of four new meroterpenoid antibiotics, merochlorins A-D, produced by the marine bacterium Streptomyces sp. strain CNH-189, which possess novel chemical skeletons unrelated to known bacterial agents. Draft genome sequencing, mutagenesis, and heterologous biosynthesis in the genome-minimized model actinomycete Streptomyces coelicolor provided the 57.6 kb merochlorin gene cluster that contains two genes encoding rare bacterial vanadium-dependent haloperoxidase (VHPO) genes. Pathway expression of two different fosmid clones that differ largely by the presence or absence of the VHPO gene mcl40 resulted in the differential biosynthesis of merochlorin C, suggesting that Mcl40 catalyzes an unprecedented 15-membered chloronium-induced macrocyclization reaction converting merochlorin D to merochlorin C.

A Stereoselective Vanadium-Dependent Chloroperoxidase in Bacterial Antibiotic Biosynthesis

Halogenases catalyze reactions that introduce halogen atoms into electron-rich organic molecules. Vanadium-dependent haloperoxidases are generally considered to be promiscuous halogenating enzymes that have thus far been derived exclusively from eukaryotes, where their cellular function is often disputed. We now report the first biochemical characterization of a bacterial vanadium-dependent chloroperoxidase, NapH1 from Streptomyces sp. CNQ-525, which catalyzes a highly stereoselective chlorination-cyclization reaction in napyradiomycin antibiotic biosynthesis. This finding biochemically links a vanadium chloroperoxidase to microbial natural product biosynthesis.

Structural models of vanadate-dependent haloperoxidases, their reactivity, immobilization on polymer support and catalytic activities

The design of structural and functional models of enzymes vanadate-dependent haloperoxidases (VHPO) and the isolation and/or generation of species having {VO(H2O)}, {VO2}, {VO(OH)} and {VO(O2)} cores, proposed as intermediate(s) during catalytic action, in solution have been studied. Catalytic potential of these complexes have been tested for oxo-transfer as well as oxidative bromination and sulfide oxidation reactions. Some of the oxidovanadium(IV) and dioxidovanadium(V) complexes have been immobilized on polymer support in order to improve their recycle ability during catalytic activities and turn over number. The formulations of the polymer-anchored complexes are based on the respective neat complexes and conclusions drawn from the various characterization studies. These catalysts have successfully been used for all catalytic reactions mentioned above. These catalysts are stable and recyclable.

V2O5 Nanowires with an Intrinsic Peroxidase‐Like Activity

AbstractV2O5 nanowires exhibit an intrinsic catalytic activity towards classical peroxidase substrates such as 2,2‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) and 3,3,5,5,‐tetramethylbenzdine (TMB) in the presence of H2O2. These V2O5 nanowires show an optimum reactivity at a pH of 4.0 and the catalytic activity is dependent on the concentration. The Michaelis‐Menten kinetics of the ABTS oxidation over these nanowires reveals a behavior similar to that of their natural vanadium‐dependent haloperoxidase (V‐HPO) counterparts. The V2O5 nanowires mediate the oxidation of ABTS in the presence of H2O2 with a turnover frequency (kcat) of 2.5 × 103 s−1. The KM values of the V2O5 nanowires for ABTS oxidation (0.4 μM) and for H2O2 (2.9 μM) at a pH of 4.0 are significantly smaller than those reported for horseradish peroxidases (HRP) and V‐HPO indicating a higher affinity of the substrates for the V2O5 nanowire surface. Based on the kinetic parameters and similarity with vanadium‐based complexes a mechanism is proposed where an intermediate metastable peroxo complex is formed as the first catalytic step. The nanostructured vanadium‐based material can be re‐used up to 10 times and retains its catalytic activity in a wide range of organic solvents (up to 90%) making it a promising mimic of peroxidase catalysts.

ChemInform Abstract: The Coordination Chemistry of Vanadium as Related to Its Biological Functions

Abstract The binding of vanadium to protein side-chains such as provided by tyrosinate, serinate, aspartate, glutamate, cysteinate, methionine and histidine is modelled by complexes with ligand sets containing phenolate, alkoxide, carboxylate, thiolate, thioether and enamine functions. The complexes mimic possible intermediates and structural motifs of the coordination environment of vanadium in vanadium–nitrogenase, vanadate-dependent haloperoxidases, the interaction with phosphorylation enzymes, and the redox-interaction with cysteinyl residues. The solid state investigations are supplemented by solution studies on vanadate–dipeptide and vanadate–adenosine systems, based on combined 51 V-NMR and potentiometric measurements. Model reactions for the function of haloperoxidases and vanadium–nitrogenase (including the alkyne–reductase and isonitrile–reductase/ligase activities) are described, and the impact of these investigations for corresponding in vitro applications is dealt with. The relevance of catecholatovanadium complexes for the accumulation of vanadium by ascidians is also addressed.

Vanadium complexes having [VIVO]2+ and [VVO2]+cores with binucleating dibasic tetradentate ligands: Synthesis, characterization, catalytic and antiamoebic activities

Binucleating hydrazones CH(2)(H(2)sal-bhz)(2) (I) and CH(2)(H(2)sal-fah)(2) (II), derived from 5,5'-methylbis(salicylaldehyde) and benzoylhydrazide or 2-furoylhydrazide, react with [V(IV)O(acac)(2)] to give dinuclear V(IV)O-complexes [CH(2){V(IV)O(sal-bhz)(H(2)O)}(2)] 1 and [CH(2){V(IV)O(sal-fah)(H(2)O)}(2)] 4, respectively. In the presence of KOH or CsOH.H(2)O, oxidation of 1 and 2 results in the formation of dioxidovanadium(v) complexes, K(2)[CH(2){V(V)O(2)(sal-bhz)}(2)].2H(2)O 2, K(2)[CH(2){V(V)O(2)(sal-fah)}(2)].2H(2)O 5, Cs(2)[CH(2){V(V)O(2)(sal-bhz)}(2)].2H(2)O 3 and Cs(2)[CH(2){V(V)O(2)(sal-fah)}(2)].2H(2)O 6. These complexes have also been prepared by aerial oxidation of in situ prepared oxidovanadium(iv) complexes 1 and 4. The compounds were characterized by IR, electronic, EPR, (1)H, (13)C and (51)V NMR spectroscopy, elemental analyses and thermogravimetric patterns. Single crystal X-ray analysis of 3 confirms the coordination of the ligand in the dianionic (ONO(2-)) enolate tautomeric form. The V(V)O(2)-complexes were used to catalyze the oxidative bromination of salicylaldehyde, therefore acting as functional models of vanadium dependent haloperoxidases, in aqueous H(2)O(2)/KBr in the presence of HClO(4) at room temperature. It is shown that the V(IV)O-complexes [CH(2){V(IV)O(sal-bhz)(H(2)O)}(2)] 1 and [CH(2){V(IV)O(sal-fah)(H(2)O)}(2)] 4 are catalyst precursors for the catalytic oxidation of organic sulfides using aqueous H(2)O(2). Plausible intermediates involved in these catalytic processes are established by UV-Vis, EPR and (51)V NMR studies. The vanadium complexes along with ligands I and II are also screened against HM1:1MSS strains of Entamoeba histolytica, the results showing that the IC(50) values of compounds 3 and 6 are lower than that of metronidazole. The toxicity studies against human cervical (HeLa) cancer cell line also showed that although compounds 3 and 6 are more toxic than metronidazole towards this cell line, the corresponding IC(50) values are relatively high, the cell viability therefore not being much affected.

Synthesis, structure and properties of three new oxidovanadium complexes containing a tridentate salicylaldehydeglycine

Three new oxidovanadium complexes as potential functional model vanadium-dependent haloperoxidases: [VO 2(C 9H 7NO 3)](C 10H 10N 2) 0.5 ( 1), [VO(C 10H 8N 2)(C 9H 7NO 3)] 3 ( 2) and [VO(C 12H 8N 2)(C 9H 7NO 3)]·2.33H 2O ( 3) have been designed and synthesized under room temperature with mixed solution of CH 3OH and H 2O. All the complexes have been characterized by elemental analysis, IR spectra, UV–vis spectroscopy and X-ray single crystal diffraction. The structural analysis indicates that the three complexes are all containing a tridentate amino-Schiff-base (salicylideneglycinate) with imine nitrogen, phenoxyl, and carboxyl oxygen as three donor atoms. The asymmetric unit of complex 1 is formed by one [VO 2(C 9H 7NO 3)] – anion and half of (C 10H 10N 2) 2+ cation, in which the nitrogen atoms of 4,4′-bipy were protonated. For complex 1, the coordination environment of the central vanadium atom is almost ideal square pyramid, while for complexes 2 and 3, the vanadium atom is a six-coordinated and in a distorted octahedral environment. It is found that there are a variety of inter- and intra-molecular hydrogen bonds in all the three complexes. What is intriguing to us is that the oxidation state of the central metal atom in complex 1 is different from that of complex 2 and 3: the oxidation state of vanadium in complex 1 is +5, while ones in complex 2 and 3 are +4. Bromination reaction activity of the complexes has been evaluated by the method with phenol red as organic substrate in the presence of H 2O 2, Br – and phosphate buffer, indicating that they can be considered as potential functional model vanadium-dependent haloperoxidases. In addition, thermal analysis, cyclic voltammetry and quantum chemistry calculations were also performed and discussed in detail.

Exploring the Chemistry and Biology of Vanadium-dependent Haloperoxidases

Nature has developed an exquisite array of methods to introduce halogen atoms into organic compounds. Most of these enzymes are oxidative and require either hydrogen peroxide or molecular oxygen as a cosubstrate to generate a reactive halogen atom for catalysis. Vanadium-dependent haloperoxidases contain a vanadate prosthetic group and utilize hydrogen peroxide to oxidize a halide ion into a reactive electrophilic intermediate. These metalloenzymes have a large distribution in nature, where they are present in macroalgae, fungi, and bacteria, but have been exclusively characterized in eukaryotes. In this minireview, we highlight the chemistry and biology of vanadium-dependent haloperoxidases from fungi and marine algae and the emergence of new bacterial members that extend the biological function of these poorly understood halogenating enzymes.

A series of oxo-vanadium(IV) complexes containing mixed ligands of poly(pyrazolyl)borate and organic carboxylic acid: Synthesis, structural characterization and primary study of bromination reaction activities

A series of oxo-vanadium(IV) complexes: Tp ∗VO(pzH ∗)(CH 3COO) ( 1), Tp ∗VO(pzH ∗)(CCl 3COO) ( 2), Tp ∗VO(pzH ∗)(C 6H 5COO) ( 3), Tp ∗VO(pzH ∗)( m-NO 2–C 6H 4COO)·CH 3CN ( 4) and [Tp ∗VO(pzH ∗)(H 2O)] +[ m-NO 2–C 6H 4–SO 3] −·CH 3OH ( 5) (Tp ∗ = hydrotris(3,5-dimethylpyrazolyl)borate; pzH ∗ = 3,5-dimethylpyrazole) are synthesized in methanol solution under physiological conditions. They are characterized by elemental analysis, IR, UV–Vis and X-ray crystallography. Structural analyses show that the vanadium atoms in complexes 1– 5 are all in a distorted-octahedral environment with the N 4O 2 donor set, and intra- or inter-hydrogen bonding linkages have been also observed in each complex. Bromination reaction activity of the complexes has been evaluated by the method with phenol red as organic substrate in the presence of H 2O 2, Br − and phosphate buffer, indicating that they can be considered as potential functional model vanadium-dependent haloperoxidases. In addition, thermal analysis and quantum chemistry calculations were also performed and discussed in detail.

Patterns of gene expression induced by oligoguluronates reveal conserved and environment-specific molecular defense responses in the brown alga Laminaria digitata.

* Until now, no information has been available on the transcriptional response to the transduction of defense signals in brown seaweeds that leads to active resistance against pathogens or grazers. * Using a polymerase chain reaction (PCR)-based, subtractive cDNA approach combined with filter arrays we demonstrated that Laminaria digitata exhibits a rapid response to oligoguluronate elicitors. The transcription levels of several genes were validated by quantitative real-time reverse-transcription PCR and further analysed using pharmacological approaches. * Fifty upregulated genes were identified by differential screening in elicited algae over a 24-h time-course. These genes were related to oxidative stress responses, production of antimicrobial secondary compounds or cell wall strengthening. Moreover, pharmacological tests showed that intracellular signal transduction is likely to involve reactive oxygen species. A new oligoguluronate-inducible vanadium-dependent haloperoxidase (vHPO), specific to iodide was also characterized. The transcription of several vHPO genes was shown to be tightly regulated. * Taken together, our data show that early transcriptional defense responses in L. digitata are similar to those in land plants but also include novel defense responses that involve tightly regulated iodine metabolism.

Preface

Knowledge of the chemistry and biochemistry of vanadium has increased enormously since the early 1980s, particularly that of vanadium(III), (IV), and (V). This led to remarkable developments in the understanding and recognition of the properties of vanadium compounds. A consequence of these new insights has been numerous applications of vanadium complexes ranging from therapeutics to catalysis and from new materials to green chemistry. Many of the more recent advances were presented at the 6th International Vanadium Symposium held 17‚Äì19 July 2008 in Lisbon, Portugal.The conference included 56 oral communications and 52 poster presentations. The following 12 papers are a selection from the oral communications presented. This conference attracted over 100 participants from 27 countries and 4 continents. The inorganic chemistry of vanadium, application of vanadium chemistry in catalysis and organic synthesis, and biological aspects of vanadium chemistry were discussed in invited lectures as well as in poster communications.The Vanadium Award, a prize first introduced at the 4th International Vanadium Symposium held in 2004 in Szeged, Hungary to recognize an outstanding contributor to the advancement of vanadium science, was awarded in 2008 to Prof. Toshikazu Hirao of the Osaka University, Japan, and his contribution is the first paper presented herein.The additional contributions that embody this issue are mostly papers covering aspects related to catalytic applications of vanadium compounds, including their use as functional models of vanadiumdependent haloperoxidases. Electron transfer in oxo and non-oxo vanadium complexes, the use of NMR to characterize the complexes, and synthesis of new polyoxovanadates are the subjects of other papers included.We look forward to the 7th International Vanadium Symposium to be held in Japan in 2010, where additional studies on the role of vanadium in life, the vanadium nutritional essentiality, vanadium toxicity, and vanadium therapy, as well as new contributions to the use of vanadium compounds in catalysis, including "green chemical" industrial applications, will certainly be presented.João Costa PessoaConference Chair

Elucidating the Protonation Site of Vanadium Peroxide Complexes and the Implications for Biomimetic Catalysis

Coordination complexes of vanadium(5+) played a key role in understanding the structure and mechanism of vanadium-dependent haloperoxidases, particularly the effects of protonation on peroxide coordination to dioxovanadium(5+) species, and in the activation of the peroxo-oxovanadium(5+) complex for substrate oxidation. There has been no spectroscopic evidence that could test the presence of a hydroxo intermediate in a catalytically active oxovanadium(5+) complex. Herein we report the use of the pre-edge transition in X-ray absorption spectroscopy as a spectroscopic signature for V=O bonding. Displacement of oxo donors with hydrogen peroxide or chloride donors dramatically decreases the pre-edge intensity, confirming that the source of the intense pre-edge feature is closely related to the -bonding associated with the V=O. Protonation of a catalytically active tripodal amine oxovanadium(5+) complex has no affect on the pre-edge intensity and, therefore, rules out the possibility of a hydroxo intermediate in the catalytic cycle.

Is vanadium a more versatile target in the activity of primordial life forms than hitherto anticipated?

The two predominant forms of vanadium occurring in the geo-, aqua- and biosphere, soluble vanadate(V) and insoluble oxovanadium(IV) (vanadyl), are subject to bacterial activity and transformation. Bacteria belonging to genera such as Shewanella, Pseudomonas and Geobacter can use vanadate as a primary electron acceptor in dissimilation or respiration, an important issue in the context of biomineralisation and soil detoxification. Azotobacter, which can employ vanadium as an essential element in nitrogen fixation, secretes a vanadophore which enables the uptake of vanadium(V). Siderophores secreted by other bacteria competitively (to ferric iron) take up vanadyl and thus interfere with iron supply, resulting in bacteriostasis. The halo-alkaliphilic Thioalkalivibrio nitratireducens possibly uses vanadium as a constituent of an alternative, molybdopterin-free nitrate reductase. Marine macro-algae can generate a variety of halogenated organic compounds by use of vanadate-dependent haloperoxidases, and a molecular vanadium compound, amavadin, from Amanita mushrooms has turned out to be an efficient catalyst in oxidation reactions. The present account is a focused and critical review of the current knowledge of the interplay of bacteria and other primitive forms of life (cyanobacteria, algae, fungi and lichens) with vanadium, with the aim to provide perspectives for applications and further investigations.

The trigonal-bipyramidal NO4 ligand set in biologically relevant vanadium compounds and their inorganic models

In the present focused review, vanadate-dependent haloperoxidases and vanadate-inhibited enzymes which catalyze the hydrolysis of phosphoester bonds are addressed. In these systems, vanadate [HxVO4](3-x)(-) is covalently coordinated to the imidazolyl moiety of an active site histidine, with a geometrical arrangement close to a trigonal bipyramid. The resulting ligand set, NO4, and ligand arrangement provide peroxidase activity to the haloperoxidases and, to a certain extent, also to vanadate-inhibited phosphatases. The haloperoxidases are responsible for the oxidative halogenation of a variety of organic substrates. They are also active in other oxidation reactions relying on peroxide as the oxidant, such as the oxidative cyclizations of terpenes and, specifically, the oxygenation of (prochiral) sulfides to (chiral) sulfoxides. These functions can be modeled by vanadium complexes. Attracted interest is paid to {V(NO4)} complexes that are functional and structural models of the peroxidases. In the vanadate-inhibited phosphatases - structural analogs of the transition state in phosphoester hydrolysis by the native enzymes - the position of the axial histidine can also be taken by cysteinate or serinate, a fact which has implications for the insulin-mimetic potential of vanadate.

Different regulation of haloperoxidation during agar oligosaccharide-activated defence mechanisms in two related red algae, Gracilaria sp. and Gracilaria chilensis

The related red seaweeds Gracilaria sp. from the eastern Mediterranean and Gracilaria chilensis from Chile were similar in their enzymatic inventory for halogenation. In both species, halogenation was dependent upon H(2)O(2) and thus driven by haloperoxidases. These could be inhibited with phosphate and reversibly inhibited with azide and were therefore apparently dependent upon vanadate. Both species generated in the first line bromoform and other brominated halocarbons. Gel electrophoresis under non-denaturating conditions demonstrated that both species expressed halogenating peroxidases. Elicitation of Gracilaria sp. with agar oligosaccharides resulted in marked increases in bromination, iodination, and chlorination. Production rates of volatile halocarbons and phenol red bromination both increased by a factor of eight, presumably due to increased availability for haloperoxidases of H(2)O(2) during the oxidative burst response. Elicitation of Gracilaria sp. also triggered a release of bromide ions through DIDS-sensitive anion channels, which allowed for some bromination in bromide-free medium. However, this effect was relatively limited. By contrast, agar oligosaccharide oxidation in G. chilensis did not increase halogenation. Obviously, agar oligosaccharide oxidation does not provide sufficient amounts of hypohalous acids for such increases, because it does not deliver H(2)O(2) at the active site of vanadium-dependent haloperoxidases. These results correlate with earlier findings that the agar oligosaccharide-elicited oxidative burst controls microorganisms while agar oligosaccharide oxidation does not.

Molecular Basis for Chloronium-mediated Meroterpene Cyclization: CLONING, SEQUENCING, AND HETEROLOGOUS EXPRESSION OF THE NAPYRADIOMYCIN BIOSYNTHETIC GENE CLUSTER

Structural inspection of the bacterial meroterpenoid antibiotics belonging to the napyradiomycin family of chlorinated dihydroquinones suggests that the biosynthetic cyclization of their terpenoid subunits is initiated via a chloronium ion. The vanadium-dependent haloperoxidases that catalyze such reactions are distributed in fungi and marine algae and have yet to be characterized from bacteria. The cloning and sequence analysis of the 43-kb napyradiomycin biosynthetic cluster (nap) from Streptomyces aculeolatus NRRL 18422 and from the undescribed marine sediment-derived Streptomyces sp. CNQ-525 revealed 33 open reading frames, three of which putatively encode vanadium-dependent chloroperoxidases. Heterologous expression of the CNQ-525-based nap biosynthetic cluster in Streptomyces albus produced at least seven napyradiomycins, including the new analog 2-deschloro-2-hydroxy-A80915C. These data not only revealed the molecular basis behind the biosynthesis of these novel meroterpenoid natural products but also resulted in the first in vivo verification of vanadium-dependent haloperoxidases.

Glycine- and Sarcosine-Based Models of Vanadate-Dependent Haloperoxidases in Sulfoxygenation Reactions

Reaction of R-styreneoxide with glycine-tert-butylester yielded amino alcohols of the general formula NR1R2R3, where R1 = CH2COOtBu and R2 = R3 = 2-phenyl-2-hydroxyethyl (H2LA); R2 = 2-phenyl-2-hydroxyethyl and R3 = 1-phenyl-2-hydroxyethyl (H2LB); R2 = H and R3 = 2-phenyl-2-hydroxyethyl (HLC); and R2 = H and R3 = 1-phenyl-2-hydroxyethyl (HLD). The corresponding reaction with sarcosine-tert-butylester and subsequent hydrolysis provided the zwitterion +NH(CH3){CH2CHPh(OH)}(CH2CO2-), HLE* (asterisk refers to unprotected carboxylate). Reaction of these ligands with VO(OiPr)3 in CH2Cl2 gave the oxovanadium(V) complexes [VOL(OiPr)2] and [VOL2(OiPr)] (for LC and LD) or, when reacted in the presence of MeOH, [VOL'(OMe)], where L' represents the methyl ester of LA, LB, and LE. The crystal and molecular structures of R-HLC, S-HLD, R,S-HLE* x H2O, and lambda-[VO(R,S-LB')OMe] have been determined. The complex [VOLB'(OMe)] contains vanadium in a distorted trigonal-bipyramidal array (tau = 0.72), the oxo group in the equatorial plane, and methoxide and N in the apical positions, and thus, it structurally models the active center of vanadate-dependent haloperoxidases. The structure and the bonding parameters, including a particularly long d(V-N) of 2.562 A, are backed up by DFT calculations. The isolated oxovanadium(V) complexes and the in situ systems L + VO(OiPr)3 catalyze the oxidation, by cumylhydroperoxide HO2R', of prochiral sulfides (MeSPh, MeSp-Tol, PhSBn) to chiral sulfoxides plus some sulfone. The best results with respect to enantioselectivity (enantiomeric excess (ee) = 38%) were obtained with the system VO(OiPr)3/LA, and the best selectivity with respect to sulfoxide (100%) was obtained with [VOLA(OiPr)]. The reaction with the hexacoordinated [VO(OMe)(HOMe)LD*] was very slow. Oxidation of PhSBn is faster than that of MeSPh and MeSpTol. Turn-over numbers are up to 60 mol of sulfoxide mol-1 of catalyst h-1 (-20 degrees C). The unspectacular ee apparently is a consequence of flexibility of the active catalyst in solution, as shown by the 51V NMR of the catalysts [VOL(OR)] and the oxo-peroxo intermediates [VOL(O2R')]. As shown by DFT calculations, the peroxo ligand coordinates in the tilted end-on fashion in the axial or equatorial position (energy difference = 17.6 kJ/mol).