The flavin-dependent halogenases are the most prevalent class of halogenase responsible for the regio- and stereoselective incorporation of halogens into natural products. These enzymes require FAD, O2, NADH, and a flavin reductase to perform catalysis. The majority of these enzymes are observed to catalyze the halogenation of aromatic substrates. CmlS is a unique member of the flavin-dependent halogenase family in that it dichlorinates an alkyl group prior to incorporation onto the antibiotic chloramphenicol. CmlS from the chloramphenicol producing strain Streptomyces venezuelae was cloned and heterologously expressed in Escherichia coli in high yield. The X-ray crystal structure of CmlS was solved to 2.2 Å resolution, yielding key insights into a potential mechanism of catalysis. As well, a covalent bond between D277 and the FAD 8α methyl group was discovered. This is a novel post-translational modification that is likely conserved among a large subset of flavin dependent halogenases and raises interesting questions about the catalytic mechanism of flavin dependent halogenation. Another key enzyme involved in the incorporation of the dichloroacetyl moiety into chloramphenicol is CmlK, an acyl-CoA synthetase. Only through the tandem action of CmlS and CmlK enzymes will chloramphenicol be formed with its dichloroacetyl group. A variety of activity assays were performed with CmlS and CmlK with the hope of discovering their substrate specificities. However, in vitro reconstitution of the activity of CmlS or CmlK was not successful. Future studies are discussed which will hopefully lead to delineating the roles of CmlS and CmlK in the biosynthesis of the dichloroacetyl group, and ultimately a detailed mechanistic description of halogenation by CmlS. / Thesis (Master, Chemistry) -- Queen's University, 2010-07-22 16:39:52.059

No description available.

Actinoplanes sp. ATCC 33002 produziert ein fünffach chloriertes Phenylpyrrolderivat, das Pentachlorpseudilin[1], für dessen Biosynthese die Beteiligung FADH2-abhängiger Halogenasen vermutet wurde. Ziel dieser Arbeit war es deshalb, in Actinoplanes sp. nach Genen FADH2-abhängiger Halogenasen zu suchen und diese heterolog zu überexprimieren. Durch Hybridisierungsexperimente mit prnC[2] (Halogenasegen aus Pseudo¬monas fluorescens) als Sonde konnten in Actinoplanes sp. ATCC 33002 zwei potentielle Gene FADH2-abhängiger Halogenasen (halA und halB) detektiert werden. Beide Gene weisen hohe Homologien zu Genen bereits bekannter FADH2-abhängiger Halogenasen auf. Die für HalA und HalB abgeleiteten Aminosäuresequenzen weisen die größten Ähnlichkeiten zur Chlortetra¬cyclin¬halogenase (Cts4)[3] aus Streptomyces aureofaciens und zur Halogenase PrnC[2] aus Pseudomonas fluorescens BL915 auf, welche im Bereich von 55 % bis 61 % liegen. HalA und HalB enthalten die in bekannten FADH2-abhängigen Halogenasen konservierten Sequenzmotive: die FADH2-Bindestelle und das Tryptophanmotiv[4]. Während die Überexpression von halA und halB in Escherichia coli zu Inclusionbodies führte, konnte halB in zwei Pseudomonadenstämmen in löslicher Form überexprimiert werden. In P. aureofaciens pCIBhalB wurde halB und in P. fluorescens pCIBhalBhis halBhis überexprimiert. In Enzymaktivitätstests mit verschiedenen Phenylpyrrolderivaten als potentiellen Substraten wurde im HalB enthaltenden Rohextrakt des Expressionsstammes P. aureofaciens pCIBhalB in vitro chlorierende Aktivität nachgewiesen. HalB gehört somit zur Gruppe der FADH2-abhängigen Halogenasen. [1] Cavalleri, B., Volpe, G., Tuan, G., Berti, M., Parenti, F., Curr. Microbiol. 1 (1978) 319 [2] Hammer, P. E., Hill, D. S., Lam, S. T., van Pée, K.-H., Ligon, J. M., Appl. Environ. Microbiol. 63 (1997) 2147 [3] Dairi, T., Nakano, T., Aisaka, K., Katsumata, R., Hasegawa, M., Biosci. Biotechnol. Biochem. 59 (1995) 1099 [4] van Pée, K.-H., Zehner, S., Enzymology and molecular genetics of biological halogenation. In: G. W. Gribble (Ed.), The Handbook of Environmental Chemistry, Vol. 3, part P. Natural production of organohalogen compounds. Springer-Verlag Berlin, Heidelberg, (2003) 171

Regioselektive Halogenasen sind Enzyme, die für die Biosynthese verschiedener halogenierter Metaboliten verantwortlich sind. Der Stamm Streptomyces rugosporus produziert die bioaktive halogenierte Verbindung Pyrroindomycin B. In dieser Arbeit wurde ein Gen einer Tryptophan-5-Halogenase aus dem Pyrroindomycinproduzenten Streptomyces rugosporus isoliert. Durch gezielte Mutation des Gens konnte die Beteiligung der Halogenase an der Biosynthese von Pyrroindomycin B nachgewiesen werden. Das Tryptophan-5-Halogenase-Gen wurde heterolog exprimiert. In einem spezifischen Enzymtest konnte die Aktivität der Tryptophan-5-Halogenase gezeigt werden. Tryptophan wurde im Enzymtest durch dieses Enzym monobromiert und monochloriert. Das Protein konnte über Nickelaffinitäts-Chromatographie gereinigt werden. / Regioselectively acting halogenases are enzymes which are responsible for the biosynthesis of a large variety of halogenated secondary metabolites. In many cases the biological activity of these metabolites is influenced by the halogen substituents. Isolation and characterization of the genes of specific halogenases and understanding the biochemistry of these enzymes are prerequisites for genetic engineering aiming at the production of novel halogenated compounds by combinatorial biosynthesis. In the work presented here a halogenase with novel regioselectivity was isolated. It is one of only three specific halogenases for which the enzymatic activity and the involvement in the biosynthesis of a halogenated metabolite could be shown. The availability of this specific tryptophan 5-halogenase is expected to open up novel possibilities for combinatorial biosynthesis and the enzymatic production of halogenated compounds.

Actinoplanes sp. ATCC 33002 produziert ein fünffach chloriertes Phenylpyrrolderivat, das Pentachlorpseudilin[1], für dessen Biosynthese die Beteiligung FADH2-abhängiger Halogenasen vermutet wurde. Ziel dieser Arbeit war es deshalb, in Actinoplanes sp. nach Genen FADH2-abhängiger Halogenasen zu suchen und diese heterolog zu überexprimieren. Durch Hybridisierungsexperimente mit prnC[2] (Halogenasegen aus Pseudo¬monas fluorescens) als Sonde konnten in Actinoplanes sp. ATCC 33002 zwei potentielle Gene FADH2-abhängiger Halogenasen (halA und halB) detektiert werden. Beide Gene weisen hohe Homologien zu Genen bereits bekannter FADH2-abhängiger Halogenasen auf. Die für HalA und HalB abgeleiteten Aminosäuresequenzen weisen die größten Ähnlichkeiten zur Chlortetra¬cyclin¬halogenase (Cts4)[3] aus Streptomyces aureofaciens und zur Halogenase PrnC[2] aus Pseudomonas fluorescens BL915 auf, welche im Bereich von 55 % bis 61 % liegen. HalA und HalB enthalten die in bekannten FADH2-abhängigen Halogenasen konservierten Sequenzmotive: die FADH2-Bindestelle und das Tryptophanmotiv[4]. Während die Überexpression von halA und halB in Escherichia coli zu Inclusionbodies führte, konnte halB in zwei Pseudomonadenstämmen in löslicher Form überexprimiert werden. In P. aureofaciens pCIBhalB wurde halB und in P. fluorescens pCIBhalBhis halBhis überexprimiert. In Enzymaktivitätstests mit verschiedenen Phenylpyrrolderivaten als potentiellen Substraten wurde im HalB enthaltenden Rohextrakt des Expressionsstammes P. aureofaciens pCIBhalB in vitro chlorierende Aktivität nachgewiesen. HalB gehört somit zur Gruppe der FADH2-abhängigen Halogenasen. [1] Cavalleri, B., Volpe, G., Tuan, G., Berti, M., Parenti, F., Curr. Microbiol. 1 (1978) 319 [2] Hammer, P. E., Hill, D. S., Lam, S. T., van Pée, K.-H., Ligon, J. M., Appl. Environ. Microbiol. 63 (1997) 2147 [3] Dairi, T., Nakano, T., Aisaka, K., Katsumata, R., Hasegawa, M., Biosci. Biotechnol. Biochem. 59 (1995) 1099 [4] van Pée, K.-H., Zehner, S., Enzymology and molecular genetics of biological halogenation. In: G. W. Gribble (Ed.), The Handbook of Environmental Chemistry, Vol. 3, part P. Natural production of organohalogen compounds. Springer-Verlag Berlin, Heidelberg, (2003) 171

Halogenases are enzymes with the ability to regioselectively and stereoselectively form carbon-halogen bonds, transferring a halogen onto various carbon scaffolds forming organohalogens. These organohalogens have many biological properties, for example, antibacterial, antifungal, anti-inflammatory, anti-proliferative, anti-fouling, anti-feedant, cytotoxic, ichthyotoxic and insecticidal activity. Additionally, the halogen is highly important for biological activity and consequently pharmaceutical and agrochemical industries are interested in environmentally sustainable and economically viable methods to selectively halogenate various organic scaffolds used during organic synthesis. One such method is to use nonheme iron halogenases, which are structurally and biochemically similar to nonheme iron hydroxylases. Common to both groups is the reactive intermediate, the iron(IV)-oxo, which abstracts a hydrogen atom from a substrate. Post hydrogen atom abstraction the catalytic cycle bifurcates, producing either hydroxylated or halogenated products. Of current debate are the factors separating halogenation and hydroxylation and in this thesis we have investigated the mechanisms of the nonheme iron halogenase (HctB) and hydroxylase (P4H) using a combination of density functional theory (DFT) and quantum mechanics/molecular mechanics (QM/MM) to gain further insight into the bifurcation factors. The QM/MM and DFT studies on the hectochlorin biosynthesis enzyme HctB revealed that substrate binding and positioning are key for optimal substrate halogenation. Additionally, key residues (Glu223) were found to influence the charge density on the chloride ligand pushing the mechanism toward halogenation. Furthermore, the influence of substrate binding and positioning was explored further in a QM/MM and MD study on the nonheme iron hydroxylase, P4H, which hydroxylates proline residues to produce 4-hydroxyproline. The QM/MM and MD study identified that mutations to either Trp243 or Tyr140 disrupted both long and short-range interactions resulting in alterations to the enzymes regioselectivity and stereoselectivity. This study also revealed that Arg161 and Glu127 formed key interactions with the substrate, which became the focus of the next study on P4H. Together these two studies on P4H, highlighted the importance of substrate positioning and selective hydrogen bonding between the protein and substrate for correct product outcome. Additionally, we were able to explore several mutations to Trp243, Tyr140, Arg161 and Glu127, identifying mutations which resulted in changes to the enzyme’s regioselectivity and stereoselectivity. Finally, in this thesis we also investigated the ability of a nonheme iron halogenase to transfer groups other than a halogen, such as nitrate and azide, using the biomimetic system , [FeIV(O)(TPA)X]+, TPA = tris(2-pyridylmethy1)amine whereby X = Cl, NO2, N3. The reaction of TPA with ethyl benzene revealed that the product distributions vary with the nature of the equatorial ligand at the metal centre. The results of this study also predict the effect of other substituents potentially opening up the application of halogenases to transferring groups other than halogens. Altogether, the studies in this thesis have looked at the different factors influencing substrate halogenation from various perspectives and have revealed the fascinating biochemistry of these enzyme’s and models to perform regioselective and stereoselective reactions with potential future industrial application.

Regioselektive Halogenasen sind Enzyme, die für die Biosynthese verschiedener halogenierter Metaboliten verantwortlich sind. Der Stamm Streptomyces rugosporus produziert die bioaktive halogenierte Verbindung Pyrroindomycin B. In dieser Arbeit wurde ein Gen einer Tryptophan-5-Halogenase aus dem Pyrroindomycinproduzenten Streptomyces rugosporus isoliert. Durch gezielte Mutation des Gens konnte die Beteiligung der Halogenase an der Biosynthese von Pyrroindomycin B nachgewiesen werden. Das Tryptophan-5-Halogenase-Gen wurde heterolog exprimiert. In einem spezifischen Enzymtest konnte die Aktivität der Tryptophan-5-Halogenase gezeigt werden. Tryptophan wurde im Enzymtest durch dieses Enzym monobromiert und monochloriert. Das Protein konnte über Nickelaffinitäts-Chromatographie gereinigt werden. / Regioselectively acting halogenases are enzymes which are responsible for the biosynthesis of a large variety of halogenated secondary metabolites. In many cases the biological activity of these metabolites is influenced by the halogen substituents. Isolation and characterization of the genes of specific halogenases and understanding the biochemistry of these enzymes are prerequisites for genetic engineering aiming at the production of novel halogenated compounds by combinatorial biosynthesis. In the work presented here a halogenase with novel regioselectivity was isolated. It is one of only three specific halogenases for which the enzymatic activity and the involvement in the biosynthesis of a halogenated metabolite could be shown. The availability of this specific tryptophan 5-halogenase is expected to open up novel possibilities for combinatorial biosynthesis and the enzymatic production of halogenated compounds.

Demosponges possess a skeleton made of a composite material with various organic constituents and/or siliceous spicules. Chitin is an integral part of the skeleton of different sponges of the order Verongida. Moreover, sponges of the order Verongida, such as Aplysina cavernicola or Ianthella basta, are well-known for the biosynthesis of brominated tyrosine derivates, characteristic bioactive natural products. It has been unknown so far whether these compounds are exclusively present in the cellular matrix or whether they may also be incorporated into the chitin-based skeletons. In the present study, we therefore examined the skeletons of A. cavernicola and I. basta with respect to the presence of bromotyrosine metabolites. The chitin-based-skeletons isolated from these sponges indeed contain significant amounts of brominated compounds, which are not easily extractable from the skeletons by common solvents, such as MeOH, as shown by HPLC analyses in combination with NMR and IR spectroscopic measurements. Quantitative potentiometric analyses confirm that the skeleton-associated bromine mainly withstands the MeOH-based extraction. This observation suggests that the respective, but yet unidentified, brominated compounds are strongly bound to the sponge skeletons, possibly by covalent bonding. Moreover, gene fragments of halogenases suggested to be responsible for the incorporation of bromine into organic molecules could be amplified from DNA isolated from sponge samples enriched for sponge-associated bacteria.

Chapter 1 Hydrocarbon autoxidation, a free radical chain reaction, is believed to play a key role in the onset and developments of most degenerative diseases and disorders. The two propagating steps: 1) H-atom abstraction from the hydrocarbon by a hydrocarbon-derived peroxyl radical, and 2) addition of oxygen to the resultant alkyl radical to form a new peroxyl, play a role in determining the rate of hydrocarbon autoxidation, as well as the regio- and stereochemistry of the product hydroperoxides. In the current study, we carried out a set of calculations to provide a detailed framework for understanding the mechanism of the first two steps of autoxidation. Chapter 2 Radical-trapping chain-breaking antioxidants inhibit hydrocarbon autoxidation. Phenols are the prototypical radical-trapping antioxidants and are employed in nature, as well as in industry, to inhibit the autoxidation of hydrocarbons. The mechanism of inhibiting radical chain propagation has recently been suggested to be a PCET on the basis of theoretical calculations. It has been demonstrated that the antioxidant activitiy of phenols is increased in the presence of either protic acids or alcohols, but the basis of this acceleration is not well understood. In the current study, we used computational methods to investigate the effects of acids and alcohols on the PCET pathway for the reaction of phenol with a peroxyl radical. Chapter 3 The antibiotic pyrrolnitrin [3-chloro-4-(2’-nitro-3’-chlorophenyl) pyrrole] (PRN) is biosynthesized from L-tryptophan in four steps, catalyzed by the enzymes PrnA, B, C and D encoded by the prn operon. Two of the four gene products, PrnA and PrnC, are flavin-dependent halogenases, a recently discovered and highly interesting class of enzymatic halogenation catalysts. Their activities have never been unequivocally demonstrated by reconstitution of the activity from a recombinant protein. Herein, we report the results of our efforts to clone the genes encoding PrnA and PrnC, and overexpress, isolate and purify the proteins from E. coli. We were able to successfully reconsistute halogenation activity of both and have obtained the first kinetic data for PrnC, which shows kinetics similar to other flavin-dependent halogenases, along with substrate inhibition. / Thesis (Master, Chemistry) -- Queen's University, 2010-02-03 15:42:39.67

No description available.