Identification of a Baeyer-Villiger monooxygenase sequence motif

Baeyer-Villiger monooxygenases (BVMOs) form a distinct class of flavoproteins that catalyze the insertion of an oxygen atom in a C-C bond using dioxygen and NAD(P)H. Using newly characterized BVMO sequences, we have uncovered a BVMO-identifying sequence motif: FXGXXXRXXXW(P/D). Studies with site-directed mutants of 4-hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB suggest that this fingerprint sequence is critically involved in catalysis. Further sequence analysis showed that the BVMOs belong to a novel superfamily that comprises three known classes of FAD-dependent monooxygenases: the so-called flavin-containing monooxygenases (FMOs), the N-hydroxylating monooxygenases (NMOs), and the BVMOs. Interestingly, FMOs contain an almost identical sequence motif when compared to the BVMO sequences: FXGXXXHXXX(Y/F). Using these novel amino acid sequence fingerprints, BVMOs and FMOs can be readily identified in the protein sequence databank. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved

Structural analysis of flavinylation in vanillyl-alcohol oxidase

Vanillyl-alcohol oxidase (VAO) is member of a newly recognized flavoprotein family of structurally related oxidoreductases. The enzyme contains a covalently linked FAD cofactor. To study the mechanism of flavinylation we have created a design point mutation (His-61 --> Thr). In the mutant enzyme the covalent His-C8 alpha -flavin linkage is not formed, while the enzyme is still able to bind FAD and perform catalysis. The H61T mutant displays a similar affinity for FAD and ADP (K-d = 1.8 and 2.1 muM, respectively) but does not interact with FMN. H61T is about 10-fold less active with 4-(methoxymethyl)phenol) (k(cat) = 0.24 s(-1), K-m = 40 muM) than the wild-type enzyme. The crystal structures of both the hole and apo form of H61T are highly similar to the structure of wild-type VAO, indicating that binding of FAD to the apoprotein does not require major structural rearrangements. These results show that covalent flavinylation is an autocatalytical process in which His-BI plays a crucial role by activating His-422. Furthermore, our studies clearly demonstrate that in VAO, the FAD binds via a typical lock-and-key approach to a preorganized binding site

Covalent flavinylation is essential for efficient redox catalysis in vanillyl-alcohol oxidase

By mutating the target residue of covalent flavinylation in vanillyl-alcohol oxidase, the functional role of the histidyl-FAD bond was studied. Three His(422) mutants (H422A, H422T, and H422C) were purified, which all contained tightly but noncovalently bound FAD. Steady state kinetics revealed that the mutants have retained enzyme activity, although the turnover rates have decreased by 1 order of magnitude. Stopped-flow analysis showed that the H422A mutant is still able to form a stable binary complex of reduced enzyme and a quinone methide product intermediate, a crucial step during vanillyl-alcohol oxidase-mediated catalysis, The only significant change in the catalytic cycle of the H422A mutant is a marked decrease in reduction rate. Redox potentials of both wild type and H422A vanillyl-alcohol oxidase have been determined. During reduction of H422A, a large portion of the neutral flavin semiquinone is observed. Using suitable reference dyes, the redox potentials for the two one-electron couples have been determined: -17 and -113 mV. Reduction of wild type enzyme did not result in any formation of flavin semiquinone and revealed a remarkably high redox potential of +55 mV, The marked decrease in redox potential caused by the missing covalent histidyl-FAD bond is reflected in the reduced rate of substrate-mediated flavin reduction limiting the turnover rate. Elucidation of the crystal structure of the H422A mutant established that deletion of the histidyl-FAD bond did not result in any significant structural changes. These results clearly indicate that covalent interaction of the isoalloxazine ring with the protein moiety can markedly increase the redox potential of the flavin cofactor, thereby facilitating redox catalysis, Thus, formation of a histidyl-EAD bond in specific flavoenzymes might have evolved as a way to contribute to the enhancement of their oxidative power

Enzymatic synthesis of vanillin

Due to increasing interest in natural vanillin, two enzymatic routes for the synthesis of vanillin were developed. The flavoprotein vanillyl alcohol oxidase (VAO) acts on a wide range of phenolic compounds and converts both creosol and vanillylamine to vanillin with high yield. The VAO-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol is further oxidized to vanillin. Catalysis is limited by the formation of an abortive complex between enzyme-bound flavin and creosol. Moreover, in the second step of the process, the conversion of vanillyl alcohol is inhibited by the competitive binding of creosol. The VAO-catalyzed conversion Of vanillylamine proceeds efficiently at alkaline pH values. Vanillylamine is initially converted to a vanillylimine intermediate product, which is hydrolyzed nonenzymatically to vanillin. This route to vanillin has biotechnological potential as the widely available principle of red pepper, capsaicin, can be hydrolyzed enzymatically to vanillylamine

Direction of the reactivity of vanillyl-alcohol oxidase with 4-alkylphenols

The covalent flavoprotein vanillyl-alcohol oxidase (VAO) predominantly converts short-chain 4-alkylphenols, like 4-ethylphenol, to (R)-1-(4'-hydroxyphenyl)alcohols and medium-chain 4-alkylphenols, like 4-butylphenol, to 1-(4'-hydroxyphenyl)alkenes. Crystallographic studies have indicated that the active site residue Asp170 is involved in determining the efficiency of substrate hydroxylation, To test this hypothesis, we have addressed the reactivity of Asp170 variants with 4-alkylphenols. The substrate preference of Asp170Glu was similar to wild type VAO, However, Asp170Ser was most active with branched-chain 4-alkylphenols. The hydroxylation efficiency of the Asp170 variants was dependent on the bulkiness of the newly introduced side chain, The Glu170 mutation favored the production of alkenes, whereas the Ser170 mutation stimulated the formation of alcohols. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved

FACTORS AFFECTING MIGRATION FROM THE CROATIAN RURAL AREA

U radu se daju rezultati istraživanja migracija u seoskom području Republike Hrvatske. Cilj je istražiti čimbenike koji utječu na iseljavanje seoskog stanovništva Republike Hrvatske. Istraživanje je provedeno 2007. godine na uzorku od 914 ispitanika dobi od 24 do 45 godina u seoskom području Republike Hrvatske. Odabir naselja i ispitanika bio je slučajan. Provedeno istraživanje pokazuje da su najveće poteškoće života u hrvatskom seoskom području gospodarske naravi, manjak zaposlenja, slaba mogućnost izbora zanimanja i niža zarada u odnosu na zaposlenje u gradu. Petina ispitanika nije zadovoljna uvjetima seoskog života i namjerava se iseliti. To je zabrinjavajući pokazatelj budući da se radi o populaciji koja je u pravilu završila proces obrazovanja i većinom osnovala obitelj. Najviše mogućih iseljenika, što je bilo i za očekivati, je iz gospodarski nerazvijenih područja Republike Hrvatske. Daljnja depopulacija hrvatskog sela bila bi pogubna, a njene najveće posljedice bile bi: prevelika urbanizacija, posebice velikih gradova, daljnji neravnomjerni razvitak Republike Hrvatske te nedovoljno iskorištenje prostornog, proizvodnog i ljudskog potencijala. S obzirom na strateški cilj ulaska Republike Hrvatske u Europsku uniju, navedeno predstavlja bitno ograničenje njene uspješne prilagodbe europskoj ekonomskoj integraciji. Iseljavanje seoskog pučanstva može se spriječiti prvenstveno povećanjem zaposlenosti i dohotka te stvaranjem takve fizičke i društvene infrastrukture u seoskom području koja će bitno poboljšati životne uvjete seoskog pučanstva. Seoska područja, poglavito gospodarski nerazvijena, nemaju dovoljno vlastitih mogućnosti za ubrzanje razvoja odnosno za nužno smanjivanje razlika u kakvoći življenja prema gradskim područjima. Zbog toga je nužno da njihov razvojni proces više nego dosada potpomogne Država osmišljenim mjerama regionalnog razvoja, uz svekoliku potporu lokalne uprave i samouprave. U tome bi svoj znatan obol trebalo dati novo-osnovano Ministarstvo za regionalni razvoj.The paper presents results of the research study on migrations in rural areas of the Republic of Croatia. The aim was to determine factors influencing migrations of rural population in Croatia. The research was carried out in 2007 on 914 respondents from 25 to 45 years of age. The rural communities and respondents were selected on a random basis. The study results indicate that the major difficulties in rural life in Croatia are of economic nature: lack of employment opportunities, inadequate choice of profession and lower income in comparison with employment in urban areas. One fifth of the respondents is not satisfied with conditions of rural life and intends to leave villages. This is a very disturbing indicator, since it refers to population, which in general, has finished education and started a family. As we expect, the largest number of potential migrants comes from economically underdeveloped Croatian areas. Further depopulation of Croatian villages would have dramatic effects, and the worst consequences would be excessive urbanization, especially of large cities, further uneven development of the Republic of Croatia, and insufficient utilization of spatial, production and human resources. Since the strategic Croatian goal is to become a member of the European Union, this is a major obstacle to its successful adjustment to the European economic integration. The migration of rural population could be prevented primarily by increase in employment and income opportunities and creation of such physical and social infrastructure in rural areas that would considerably improve living conditions for rural population. The rural areas, particularly underdeveloped, have no adequate capacities for intensification of its development and diminishing differences in their quality of life compared to urban areas. Thus, the state support is increasingly required by introducing measures of regional development with complementary support of the local government. The newly founded Ministry of Regional Development is therefore inevitable in this process

Baeyer-Villiger monooxygenases, an emerging family of flavin-dependent biocatalysts

Baeyer-Villiger monooxygenases (BVMOs) are flavoenzymes that catalyze a remarkably wide variety of oxidative reactions such as regio- and enantioselective Baeyer-Villiger oxidations and sulfoxidations. Several of these conversions are difficult to achieve using chemical approaches. Due to their selectivity and catalytic efficiency, BVMOs are highly valuable biocatalysts for the synthesis of a broad range of fine chemicals. For a long time, only one member of this class of flavin-containing biocatalysts had been cloned and overexpressed which has limited their application for synthetic processes. Recently a number of new genes that encode BVMOs have been sequenced and overexpressed. In this paper the biocatalytic properties of recently cloned BVMOs are reviewed. Furthermore, the potential for obtaining novel BVMOs from sequenced genomes will be discussed

Coenzyme binding is beneficial for the stability of 4 hydroxyacetophenone monooxygenase

The NADPH-dependent dimeric flavoenzyme 4-hydroxyacetophenone monooxygenase (HAPMO) catalyzes Baeyer-Villiger oxidations of a wide range of ketones, thereby generating esters or lactones. In the current work, we probed HAPMO-coenzyme complexes present during the enzyme catalytic cycle with the aim to gain mechanistic insight. Moreover, we investigated the structural role of the nicotinamide coenzyme. For these studies, we used (i) wild type HAPMO, (ii) the R339A variant, which is active but has a low affinity toward NADPH, and (iii) the R440A variant, which is inactive but has a high affinity toward NADPH. Electrospray ionization mass spectrometry was used as the primary tool to directly observe noncovalent protein-coenzyme complexes in real time. These analyzes showed for the first time that the nicotinamide coenzyme remains bound to HAPMO during the entire catalytic cycle of the NADPH oxidase reaction. This may also have implications for other homologous Baeyer-Villiger monooxygenases. Together with the observations that NADP+ only weakly interacts with oxidized enzyme and that HAPMO is mainly in the reduced form during catalysis, we concluded that NADP+ interacts tightly with the reduced form of HAPMO. We also demonstrated that the association with the coenzyme is crucial for enzyme stability. The interaction with the coenzyme analog 3-aminopyridine adenine dinucleotide phosphate (AADP+) strongly enhanced the thermal stability of wild type HAPMO. This coenzyme-induced stabilization may also be important for related enzymes