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Functions and Applications of Nmos

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Functions and Applications of Nmos Functions and applications of NMOs Willem Dijkman (1606212), June 20 2009 Assisted by drs. Anette Riebel and prof. dr. ir. Marco W. Fraaije Abstract The hydroxylation of amino groups is performed by different types of enzymes. Some of these types are metal dependent whereas others need a flavin cofactor. These flavoproteins (NMOs, for N-hydroxylating monooxygenases) can be assigned to subclass B of the external flavoproteins, an enzyme class consisting of enzymes with a broad range of monooxygenating activity. NMOs are mostly found in the biosynthesis of siderophores of different bacteria. Recently a NMO has been identified in the biosynthesis of a kutzneride. NMOs have a narrow substrate range, making them less applicable in industry. NMOs in siderophore biosynthesis can however be targeted to slow down bacterial growth. Contents enzymes which perform these monooxygenations often contain metal Introduction 1 atoms. Different types of enzymes are Classification of flavoproteins 2 known. P450 enzymes as well as non-heme 2 The catalytic cycle of flavins 3 monooxygenases contain iron and copper- N-hydroxylation in nature 4 dependent enzymes also perform Characteristics of NMOs 4 monooxygenations. Not all enzymes depend - NMOs in siderophore synthesis 5 on a (metallic) cofactor for their activity: - A NMO in kutzneride synthesis 7 flavin-dependent monooxygenases make use - N-hydroxylation in valanimycin synthesis 7 of a flavin group instead of a metal ion and Recombinant NMOs 8 recently enzymes without any cofactor have 3,4 Pharmaceutical applications 8 shown monooxygenase activity . Industrial applications 9 In this article one type of monooxygenation Conclusion 10 will be described, focusing on one group of Literature 10 monooxygenating enzymes. The oxidation of an amine to a hydroxylamine will be described, performed by NMOs. NMOs are Introduction flavin dependent enzymes similar to some Baeyer-Villiger monooxygenases and therefore classified to the same subclass of In nature, many enzymes with flavin dependent monooxygenases. Because monooxygenase activity are known. the wide variety of flavoproteins, an overview Monooxygenation reactions can introduce a of the subclasses of external flavin dependent wide variety of functional groups: epoxides monooxygenases will be given first. Also the are formed from carbon-carbon double different states of the flavin cofactor during bonds, esters and lactones can be formed catalysis will be discussed. After this, the role from ketones in a Baeyer-Villiger reaction, of NMOs in nature will be shown and some carbon and nitrogen atoms are hydroxylated NMOs will be discussed in detail. Substrate and heteroatoms like sulfur, phosphorus and and cofactor preferences will be mentioned as selenium can be oxidized. The reaction well is the effects of mutations on these between molecular oxygen (O2) and an enzymes. The article will be concluded with organic compound is spin forbidden and perspectives of NMOs in industrial and therefore hard to achieve pharmaceutical applications. without enzymes 1. The above mentioned conversions achieved by chemical means often require high temperatures and strong, and therefore hazardous reagents. In nature, 1 Classification of flavoproteins hydroxylating monooxygenases (NMOs). NMOs catalyze the hydroxylating of primary Many attempt have been done to make a amines mainly in the biosynthesis of satisfactory classification for different types siderophores. This article will focus on NMOs of flavoproteins, depending on the type of and therefore more information will be chemical reaction, the nature of the oxidizing provided later in the article. and reducing substrates and more recently the structural motifs determined by X-ray In the two subclasses just described, the crystallography 5. Recently a new reduction of flavin adenine dinucleotide (FAD) classification has been made, depending on is done by the same enzyme as the oxidation sequence and structural data 1. Using this of the substrate, enzymes in subclass A and classification, flavoproteins can be divided B are therefore one component systems. In into 6 classes (table 1). The first two all other subclasses, one enzyme uses subclasses (A and B) consist of one reduced flavin to incorporate an oxygen atom polypeptide chain, subclass C-F depend on in the substrate and a separate enzyme uses more components for their catalytic activity. NAD(P)H to reduce the flavin. All upcoming subclasses therefore are two component Enzymes in subclass A generally perform an systems. electrophilic attack on activated hydroxyl and amino groups on aromatic compounds. The C subclass contains enzyme complexes Members from this class have a narrow with one are two monooxygenase and one substrate range. They are usually involved in reducing component. This is the only subclass 1 the microbial degradation of aromatic in the classification of Van Berkel et al. using compounds by hydroxylation. Other subclass flavin mononucleotide (FMN) as flavin, all A flavin-dependent monooxygenases are other classes use FAD. involved in the biosynthesis of ubiqinone and The most extensively studied members of the modification of aromatic polyketides. this subclass are bacterial luciferases. These Other members of this subclass perform enzymes emit light when oxidizing long-chain epoxidations instead of hydroxylations. aliphatic aldehydes. Luciferases consist of a One well studied enzyme from this subclass is reductase component and a hetero dimeric 4-hydroxybenzoate 3-monooxygenase, a oxygenase component. This hetero dimer protein found in Pseudomonas species 6. consists of two quite similar peptide chains, Several other enzymes have been studied as but only one chain has an active site. well. Next to luciferases, Type II BVMOs (class C BVMOs) belong to this subclass. Although Enzymes in subclass B can be divided class B and C BVMOs perform the same type according to their specificity. The first group of reaction, they are different in structure and is the flavin-containing monooxygenases sequence. It is shown that luciferases can (FMOs). These enzymes can catalyze the also perform Baeyer-Villiger oxidations. In monooxygenation of carbon-bound reactive contrast, light emission during catalyzes of heteroatoms like nitrogen, phosphorus, class C BVMOs has never been reported. sulfur, selenium or iodine. FMOs are present In addition, some enzymes performing a in all eukaryotes and are important in sulfur oxidation have also been assigned to detoxification processes, in prokaryotes these this C subclass, based on similarities in enzymes are quite rare. structure and sequence. The second group in subclass B consist of Type I BVMOs, or Type I Baeyer-Villiger Subclass D members catalyze reactions on Monooxygenases, now called class B BVMOs. similar substrates to the substrates in This name clearly indicates the reaction they subclass A: aromatic compounds. But unlike perform: a Baeyer-Villiger oxidation. In this subclass A enzymes, this enzymes only reaction a ketone (or aldehyde) is converted perform hydroxylation, epoxilations are not to an ester or a lactone. Several proteins of observed (yet). Several of these enzymes are this group have been studied. involved in dechlorination. For example 4- The last group belonging to the subclass B chlorophenol is hydroxylated and thereby flavin-dependent monooxygenases is the N- dechlorinated. 2 Table 1. Classification of external flavoprotein monooxygenases by Van Berkel et al.1. Subclass Prototype Reactions Subunits Cofactor Coenzyme A 4-OH-benzoate Hydroxyaltion α FAD NAD(P)H hydroxylase epoxilation B Cyclohexanone Baeyer-Villiger; N- α FAD NADPH monooxygenase oxidation C Luciferase Light emision; α+β FMN NAD(P)H Baeyer-Villiger; S- oxidation D 4-OH-pennylacetate Hydroxylation α+β FAD NAD(P)H hydroxylase E Styrene Epoxylation α+β FAD NAD(P)H monooxygense F Tryptophan 7- Halogenation α+β FAD NAD(P)H halogenase Table taken from Van Berkel et al. 2006 1. In the column ‘Reactions’ the most commonly oxidations found in vivo are listed In subclass E only a very limited number of enzymes are found. All of them are styrene The catalytic cycle of flavins monooxygenases. The enzymes oxidize styrene and some derivates on a highly To perform this type of reactions, the enantioselective way, these oxidized enzymes needs a reduced flavin cofactor. compounds are used as building blocks in the Some enzymes use FAD whereas others use fine-chemical industry. FMN. Both molecules have the same catalytic cycle. The last subclass (F) contains enzymes which First NAD(P)H is used to convert oxidized perform halogenations. Therefore these flavin to reduced flavin. When reduced flavin enzymes are not oxygenases, but they is present it can react with molecular oxygen perform a reaction which is mechanistically giving a peroxyflavin. This flavin species can similar to the reaction of flavoprotein be converted to a hydroperoxyflavin when a monooxygenases. These halogenase enzymes proton is taken up, or the peroxyflavin can are found in biosynthetic routes for antitumor perform a nucleophilic oxygenation with a agents and antibiotics. substrate, leading to the product and hydroxyflavin. In the case of a Flavin-dependent monooxygenases form a hydroperoxyflavin an electrophilic major group and cover a wide range of oxygenation leads to the product and again oxygenation reactions. hydroxyflavin. Normally a hydroperoxy The use of these enzymes has been reported compound is unstable, and in the absence of in different areas. Styrene oxidase
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