Structural and biochemical characterization of 3-hydroxybenzoate 6-hydroxylase

The thesis deals with the characterization of a new flavoprotein hydroxylase 3 hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1. 3HB6H is able to insert exclusively oxygen in para-position and the enzyme has been chosen to study the structural basis of such regioselectivity. As main result, functional mirror image active sites direct regioselective 3-hydroxybenzoate hydroxylation. Moreover, the nature and role of unprecedented phospholipid binding has been analyzed demonstrating a role in enzyme oligomerization and a possible protective role during catalysis. To conclude, the knowledge acquired improves our insight into the strategies of flavin-dependent regioselective hydroxylation and the results emerged in this thesis provide a foundation for further structural and kinetic studies on 3HB6H and related enzymes. </p

Crystal Structure of 3-Hydroxybenzoate 6-Hydroxylase Uncovers Lipid-assisted Flavoprotein Strategy for Regioselective Aromatic Hydroxylation.

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a dimeric flavoprotein that catalyzes the NADH- and oxygen-dependent para-hydroxylation of 3-hydroxybenzoate to 2,5-dihydroxybenzoate. In this study, we report the crystal structure of 3HB6H as expressed in Escherichia coli. The overall fold of 3HB6H is similar to that of p-hydroxybenzoate hydroxylase and other flavoprotein aromatic hydroxylases. Unexpectedly, a lipid ligand is bound to each 3HB6H monomer. Mass spectral analysis identified the ligand as a mixture of phosphatidylglycerol and phosphatidylethanolamine. The fatty acid chains occupy hydrophobic channels that deeply penetrate into the interior of the substrate-binding domain of each subunit, whereas the hydrophilic part is exposed on the protein surface, connecting the dimerization domains via a few interactions. Most remarkably, the terminal part of a phospholipid acyl chain is directly involved in the substrate-binding site. Co-crystallized chloride ion and the crystal structure of the H213S variant with bound 3-hydroxybenzoate provide hints about oxygen activation and substrate hydroxylation. Essential roles are played by His-213 in catalysis and Tyr-105 in substrate binding. This phospholipid-assisted strategy to control regioselective aromatic hydroxylation is of relevance for optimization of flavin-dependent biocatalysts

Оцінювання закону розподілу величини збитків унаслідок реалізації загрози “Відсутність або недостатність технічного обслуговування”

The problem of estimation of size distribution law of damages in the absence or insufficient sample size of the universe and the initial data suggested approach to finding the most expected value of losses due to information security threats

Functional annotation and characterization of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1

The genome of Rhodococcus jostii RHA1 contains an unusually large number of oxygenase encoding genes. Many of these genes have yet an unknown function, implying that a notable part of the biochemical and catabolic biodiversity of this Gram-positive soil actinomycete is still elusive. Here we present a multiple sequence alignment and phylogenetic analysis of putative R. jostii RHA1 flavoprotein hydroxylases. Out of 18 candidate sequences, three hydroxylases are absent in other available Rhodococcus genomes. In addition, we report the biochemical characterization of 3-hydroxybenzoate 6-hydroxylase (3HB6H), a gentisate-producing enzyme originally mis-annotated as salicylate hydroxylase. R. jostii RHA1 3HB6H expressed in Escherichia coli is a homodimer with each 47 kDa subunit containing a non-covalently bound FAD cofactor. The enzyme has a pH optimum around pH 8.3 and prefers NADH as external electron donor. 3HB6H is active with a series of 3-hydroxybenzoate analogues, bearing substituents in ortho- or meta-position of the aromatic ring. Gentisate, the physiological product, is a non-substrate effector of 3HB6H. This compound is not hydroxylated but strongly stimulates the NADH oxidase activity of the enzyme -------------------------------------------------------------------------------

Evaluation of exopolysaccharide production by Leuconostoc mesenteroides strains isolated from wine

Exopolysaccharide (EPS)-producing lactic acid bacteria are responsible for the alteration of wine and other fermented beverages. The potential to produce EPS was investigated for Leuconostoc mesenteroides strains isolated from Spanish grape must and wine. Most strains were able to produce EPS from sucrose containing media. Based on their EPS-producing phenotype and on their EPSmonosaccharide composition, the L. mesenteroides strains analyzed could be arranged in 2 groups. One group comprises mucoid strains producing a glucan polymer, and the other group includes strains producing a fructan polymer. The presence of a glucosyltransferase encoding gene in the glucan producing L. mesenteroides strains was assayed by PCR. Two primer sets, PF1-PF8 and GTFFGTFR, were used to amplify internal fragment of known glucosyltransferase genes. None of the glucan-producing strains gave a positive amplicon by the primer sets used. Therefore, new tools need to be developed to broaden the range of potentially spoiling agents detected by PCR in fermented beveragesThis study was supported by grants AGL2005-00470 (CICYT), FUN-C-FOOD Consolider 25506 (MEC), RM03-002 (INIA), andS-0505/AGR/000153 (CAM). The technical assistance of M.V. Santamar ´ıa and A. Gómez is greatly appreciated. S. Montersino is a recipient of an Erasmus fellowship.Peer reviewe

The flavin monooxygenases

Flavin monooxygenases are ubiquitous enzymes that catalyze a wide variety of regio -and enantioselective oxygenation reactions via the formation of a fl avin C4a-(hydro)peroxide intermediate. Based on fold and function, fl avin monooxygenases can be divided into six subfamilies. Subclasses A and B comprise single-component enzymes that rely on NAD(P)H as electron donor. Subclasses C–F comprise two-component enzymes , composed of a fl avin reductase and a fl avin-specifi c monooxygenase . FAD-containing hydroxylases (subclass A ) convert many (hetero)aromatic substrates ranging from monophenols and uric acids to polyketide antibiotics and antitumor agents. FAD-containing monooxygenases (subclass B ) perform Baeyer-Villiger oxidation, sulfoxidation and heteroatom hydroxylation reactions. FMN-dependent TIM-barrel enzymes (subclass C ) catalyze Baeyer-Villiger oxidation, hydroxylation of long-chain alkanes and nitriloacetate, oxidation and desulfurization of sulfonates, and oxidation of aldehydes coupled with generation of bioluminescence. FMN/FAD hydroxylases with an acyl-CoA dehydrogenase fold (subclass D ) convert mono- and polyphenols. FAD-specifi c styrene monooxygenases (subclass E ) oxidize styrene derivatives to the corresponding epoxides. FAD-specifi c halogenases (subclass F ) catalyze the regioselective chlorination and bromination of activated organic molecules for the production of antibiotics, antitumor agents and other natural products. During the past few years, many new family members and several unprecedented fl avin monooxygenase reactions have emerged. This review illustrates selected features, tools to retrieve novel fl avin monooxygenases from genome mining, and new findings on each of the six subclasses

The flavin monooxygenases

Flavin monooxygenases are ubiquitous enzymes that catalyze a wide variety of regio -and enantioselective oxygenation reactions via the formation of a fl avin C4a-(hydro)peroxide intermediate. Based on fold and function, fl avin monooxygenases can be divided into six subfamilies. Subclasses A and B comprise single-component enzymes that rely on NAD(P)H as electron donor. Subclasses C–F comprise two-component enzymes , composed of a fl avin reductase and a fl avin-specifi c monooxygenase . FAD-containing hydroxylases (subclass A ) convert many (hetero)aromatic substrates ranging from monophenols and uric acids to polyketide antibiotics and antitumor agents. FAD-containing monooxygenases (subclass B ) perform Baeyer-Villiger oxidation, sulfoxidation and heteroatom hydroxylation reactions. FMN-dependent TIM-barrel enzymes (subclass C ) catalyze Baeyer-Villiger oxidation, hydroxylation of long-chain alkanes and nitriloacetate, oxidation and desulfurization of sulfonates, and oxidation of aldehydes coupled with generation of bioluminescence. FMN/FAD hydroxylases with an acyl-CoA dehydrogenase fold (subclass D ) convert mono- and polyphenols. FAD-specifi c styrene monooxygenases (subclass E ) oxidize styrene derivatives to the corresponding epoxides. FAD-specifi c halogenases (subclass F ) catalyze the regioselective chlorination and bromination of activated organic molecules for the production of antibiotics, antitumor agents and other natural products. During the past few years, many new family members and several unprecedented fl avin monooxygenase reactions have emerged. This review illustrates selected features, tools to retrieve novel fl avin monooxygenases from genome mining, and new findings on each of the six subclasses

Catalytic and structural features of flavoprotein hydroxylases and epoxidases

Monooxygenases perform chemo-, regio- and/or enantioselective oxygenations of organic substrates under mild reaction conditions. These properties and the increasing number of representatives along with effective preparation methods place monooxygenases in the focus of industrial biocatalysis. Mechanistic and structural insights reveal reaction sequences and allow turning them into efficient tools for the production of valuable products. Herein we describe two biocatalytically relevant subclasses of flavoprotein monooxygenases with a close evolutionary relation: subclass A represented by p-hydroxybenzoate hydroxylase (PHBH) and subclass E formed by styrene monooxygenases (SMOs). PHBH family members perform highly regioselective hydroxylations on a wide variety of aromatic compounds. The more recently discovered SMOs catalyze a number of stereoselective epoxidation and sulfoxidation reactions. Mechanistic and structural studies expose distinct characteristics, which provide a promising source for future biocatalyst development