The Thioesterase Bhp is Involved in the Formation of β-Hydroxytyrosine during Balhimycin Biosynthesis in Amycolatopsis balhimycina

The putative hydrolase gene bhp from the balhimycin biosynthetic gene cluster has been cloned and overexpressed in Escherichia coli. The corresponding enzyme Bhp was purified to homogeneity by nickel-chelating chromatography and characterized. Although Bhp has sequence similarities to hydrolases with “haloperoxidase”/perhydrolase activity, it did not show any enzymatic activity with standard “haloperoxidase”/perhydrolase substrates (e.g., monochlorodimedone and phenol red), nonspecific esterase substrates (such as p-nitrophenyl acetate, p-nitrophenyl phosphate and S-thiophenyl acetate) or the model lactonase substrate dihydrocoumarin. However, Bhp could be shown to catalyse the hydrolysis of S-β-hydroxytyrosyl-N-acetyl cysteamine thioester (β-OH-Tyr-SNAC) with 15 times the efficiency of S-L-tyrosyl- N-acetyl cysteamine thioester (L-Tyr-SNAC). This is in agreement with the suggestion that Bhp is involved in balhimycin biosynthesis, during which it was supposed to catalyse the hydrolysis of β-OH-Tyr-S-PCP (PCP=peptidyl carrier protein) to free β-hydroxytyrosine (β-OH-Tyr) and strongly suggests that Bhp is a thioesterase with high substrate specificity for PCP-bound β-OH-Tyr and not a “haloperoxidase”/perhydrolase or nonspecific esterase

Dissecting the low catalytic capability of flavin-dependent halogenases

Although flavin-dependent halogenases (FDHs) are attractive biocatalysts, their practical applications are limited because of their low catalytic efficiency. Here, we investigated the reaction mechanisms and structures of tryptophan 6-halogenase (Thal) from Streptomyces albogriseolus using stopped-flow, rapid-quench flow, quantum/mechanics molecular mechanics calculations, crystallography, and detection of intermediate (hypohalous acid [HOX]) liberation. We found that the key flavin intermediate, C4a-hydroperoxyflavin (C4aOOH-FAD), formed by Thal and other FDHs (tryptophan 7-halogenase [PrnA] and tryptophan 5-halogenase [PyrH]), can react with I-, Br-, and Cl- but not F- to form C4a-hydroxyflavin and HOX. Our experiments revealed that I- reacts with C4aOOH-FAD the fastest with the lowest energy barrier and have shown for the first time that a significant amount of the HOX formed leaks out as free HOX. This leakage is probably a major cause of low product coupling ratios in all FDHs. Site-saturation mutagenesis of Lys79 showed that changing Lys79 to any other amino acid resulted in an inactive enzyme. However, the levels of liberated HOX of these variants are all similar, implying that Lys79 probably does not form a chloramine or bromamine intermediate as previously proposed. Computational calculations revealed that Lys79 has an abnormally lower pKa compared with other Lys residues, implying that the catalytic Lys may act as a proton donor in catalysis. Analysis of new X-ray structures of Thal also explains why premixing of FDHs with reduced flavin adenine dinucleotide generally results in abolishment of C4aOOH-FAD formation. These findings reveal the hidden factors restricting FDHs capability which should be useful for future development of FDHs applications.</p

An unusual flavin-dependent halogenase from the metagenome of the marine sponge Theonella swinhoei WA

The authors thank EU BlueGenics (Seventh Framework Programme, Collaborative project “BlueGenics”, Grant no. 311848 RJMG and JP), the SNF (Grant no.205321_165695 to JP), the Helmut Horten Foundation (JP), and ERAIB (Grant no. 031A338A KHVP and RJMG) for funding.Uncultured bacteria from sponges have been demonstrated to be responsible for the generation of many potent, bioactive natural products including halogenated metabolites.1 The identification of gene clusters from the metagenomes of such bacterial communities enables the discovery of enzymes that mediate new and useful chemistries and allows insight to be gained into the biogenesis of potentially pharmacologically important natural products. Here we report a new pathway to the keramamides (krm); the first functional evidence for the existence of a distinct producer in the Theonella swinhoei WA chemotype is revealed, and a key enzyme on the pathway, a unique flavin dependent halogenase with a broad substrate specificity, and with potential as a useful new biocatalytic tool is described.PostprintPeer reviewe

A role for the cell-wall protein silacidin in cell size of the diatom Thalassiosira pseudonana

Diatoms contribute 20% of global primary production and form the basis of many marine food webs. Although their species diversity correlates with broad diversity in cell size, there is also an intraspecific cell-size plasticity due to sexual reproduction and varying environmental conditions. However, despite the ecological significance of the diatom cell size for food-web structure and global biogeochemical cycles, our knowledge about genes underpinning the size of diatom cells remains elusive. Here, a combination of reverse genetics, experimental evolution and comparative RNA8 sequencing analyses enabled us to identify a previously unknown genetic control of cell size in the diatom Thalassiosira pseudonana. In particular, the targeted deregulation of the expression of the cell-wall protein silacidin caused a significant increase in valve diameter. Remarkably, the natural downregulation of the silacidin gene transcript due to experimental evolution under low temperature also correlated with cell-size increase. Our data give first evidence for a genetically controlled regulation of cell size in Thalassiosira pseudonana and possibly other centric diatoms as they also encode the silacidin gene in their genomes

Cloning and high-level expression of a chloroperoxidase gene from Pseudomonas pyrrocinia in Escherichia coli

AbstractA chloroperoxidase gene from Pseudomonas pyrrocinia was cloned into Escherichia coli using the cosmid vector pJB8. The gene coding for the chloroperoxidase could be localized to a 1.5 kb fragment of DNA which was subcloned into the high-copy-number plasmid pUC18. In one subclone increased halogenating activity could be found which was 570-fold greater than in P. pyrrocinia. The halogenating enzyme was identified as the chloroperoxidase by SDS-polyacrylamide gel electrophoresis

Crystallization and X-ray diffraction of a halogenating enzyme, tryptophan 7-halogenase, from Pseudomonas fluorescens

Chlorination of natural products is often required for their biological activity; notable examples include vancomycin, the last-ditch antibiotic. It is now known that many chlorinated natural products are made not by haloperoxidases, but by FADH2-dependent halogenases. The mechanism of the flavin-containing enzymes is obscure and there are no structural data. Here, crystals of PrnA (tryptophan 7-halogenase), an enzyme that regioselectively chlorinates tryptophan, cocrystallized with tryptophan and FAD are reported. The crystals belong to the tetragonal space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 67.8, c = 276.9 A. A data set to 1.8 A with 93% completeness and an Rmerge of 7.1% has been collected from a single flash-cooled crystal. A method for incorporating selenomethionine in a Pseudomonas fluorescens expression system also is reported

Two-Component FAD-Dependent Monooxygenases: Current Knowledge and Biotechnological Opportunities

Flavoprotein monooxygenases create valuable compounds that are of high interest for the chemical, pharmaceutical, and agrochemical industries, among others. Monooxygenases that use flavin as cofactor are either single- or two-component systems. Here we summarize the current knowledge about two-component flavin adenine dinucleotide (FAD)-dependent monooxygenases and describe their biotechnological relevance. Two-component FAD-dependent monooxygenases catalyze hydroxylation, epoxidation, and halogenation reactions and are physiologically involved in amino acid metabolism, mineralization of aromatic compounds, and biosynthesis of secondary metabolites. The monooxygenase component of these enzymes is strictly dependent on reduced FAD, which is supplied by the reductase component. More and more representatives of two-component FAD-dependent monooxygenases have been discovered and characterized in recent years, which has resulted in the identification of novel physiological roles, functional properties, and a variety of biocatalytic opportunities

Tryptophan 7-halogenase (PrnA) structure suggests a mechanism for regioselective chlorination

Chlorinated natural products include vancomycin and cryptophycin A. Their biosynthesis involves regioselective chlorination by flavin-dependent halogenases. We report the structural characterization of tryptophan 7-halogenase (PrnA), which regioselectively chlorinates tryptophan. Tryptophan and flavin adenine dinucleotide (FAD) are separated by a 10 angstrom-long tunnel and bound by distinct enzyme modules. The FAD module is conserved in halogenases and is related to flavin-dependent monooxygenases. On the basis of biochemical studies, crystal structures, and by analogy with monooxygenases, we predict that FADH2 reacts with O2 to make peroxyflavin, which is decomposed by Cl-. The resulting HOCl is guided through the tunnel to tryptophan, where it is activated to participate in electrophilic aromatic substitution

Characterization of Two Hydrogen Peroxide Resistant Peroxidases from Rhodococcus opacus 1CP

The dye-decolorizing peroxidases (DyP) are a family of heme-dependent enzymes present on a broad spectrum of microorganisms. While the natural function of these enzymes is not fully understood, their capacity to degrade highly contaminant pigments such as azo dyes or anthraquinones make them excellent candidates for applications in bioremediation and organic synthesis. In this work, two novel DyP peroxidases from the organism Rhodococcus opacus 1CP (DypA and DypB) were cloned and expressed in Escherichia coli. The enzymes were purified and biochemically characterized. The activities of the two DyPs via 2,2′-azino-bis [3-ethylbenzthiazoline-6-sulphonic acid] (ABTS) assay and against Reactive Blue 5 were assessed and optimized. Results showed varying trends for DypA and DypB. Remarkably, these enzymes presented a particularly high tolerance towards H2O2, retaining its activities at about 10 mM H2O2 for DypA and about 4.9 mM H2O2 for DypB