A Unique Combination of Genetic Systems for the Synthesis of Trehalose in
            <i>Rubrobacter xylanophilus</i>
            : Properties of a Rare Actinobacterial TreT

Nobre, Ana; Alarico, Susana; Fernandes, Chantal; Empadinhas, Nuno; Costa, Milton S. da

doi:10.1128/jb.01055-08

Cited by 35 publications

(39 citation statements)

References 40 publications

(77 reference statements)

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“…The enzyme displayed maximum activity at 65°C (Fig. 4B), which is in good agreement with the optimum growth temperature of R. xylanophilus and similar to that of other enzymes from this bacterium (15,16). An Arrhenius plot showed that the activation energy of the reaction was approximately constant in the temperature range from 30°C to 50°C, and the activation energy was calculated as 62.1 kJ/mol (see Fig.…”

Section: Resultssupporting

confidence: 69%

“…Such differences have also been reported for other genes from R. xylanophilus (16), indicating the necessity for reanalyzing the genome sequence. Interestingly, the amino acid sequence of RxDAO showed a higher identity to hypothetical proteins of the phylogenetically distant Gram-negative bacteria S. aurantiaca and M. stipitatus than that of DAOs of phylogenetically close Gram-positive bacteria S. coelicolor and A. protophormiae.…”

Section: Discussionmentioning

confidence: 88%

“…This bacterium also exhibits extreme radiation and desiccation resistance. Several enzymes from this thermophilic bacterium, such as mannosyl-3-phosphoglycerate synthase and the enzymes involved in trehalose synthesis (trehalose-6-phosphate synthase, trehalose-6-phosphate phosphatase, and trehalose glycosyltransferring synthase), have been characterized and shown to exhibit higher thermal stability (16,17), suggesting that this organism may be a candidate source of stable enzymes.…”

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confidence: 99%

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A Highly Stable d -Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus

Takahashi

Furukawara

Omae

et al. 2014

Appl Environ Microbiol

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D-Amino acid oxidase (DAO) is a biotechnologically attractive enzyme that can be used in a variety of applications, but its utility is limited by its relatively poor stability. A search of a bacterial genome database revealed a gene encoding a protein homologous to DAO in the thermophilic bacterium Rubrobacter xylanophilus (RxDAO). The recombinant protein expressed in Escherichia coli was a monomeric protein containing noncovalently bound flavin adenine dinucleotide as a cofactor. This protein exhibited oxidase activity against neutral and basic D-amino acids and was significantly inhibited by a DAO inhibitor, benzoate, but not by any of the tested D-aspartate oxidase (DDO) inhibitors, thus indicating that the protein is DAO. RxDAO exhibited higher activities and affinities toward branched-chain D-amino acids, with the highest specific activity toward D-valine and catalytic efficiency (k cat /K m ) toward D-leucine. Substrate inhibition was observed in the case of D-tyrosine. The enzyme had an optimum pH range and temperature of pH 7.5 to 10 and 65°C, respectively, and was stable between pH 5.0 and pH 8.0, with a T 50 (the temperature at which 50% of the initial enzymatic activity is lost) of 64°C. No loss of enzyme activity was observed after a 1-week incubation period at 30°C. This enzyme was markedly inactivated by phenylmethylsulfonyl fluoride but not by thiol-modifying reagents and diethyl pyrocarbonate, which are known to inhibit certain DAOs. These results demonstrated that RxDAO is a highly stable DAO and suggested that this enzyme may be valuable for practical applications, such as the determination and quantification of branched-chain D-amino acids, and as a scaffold to generate a novel DAO via protein engineering.T he flavoenzyme D-amino acid oxidase (DAO; EC 1.4.3.3) catalyzes the oxidative deamination of neutral and basic D-amino acids but not of acidic D-amino acids, which are substrates of D-aspartate oxidase (DDO). DAO was first identified in pig kidney (pkDAO) by Krebs in 1935 (1) and subsequently has been found mainly in eukaryotic organisms, ranging from fungi to humans (2). These eukaryotic DAOs have been extensively studied as a model of the oxidase-dehydrogenase class of flavoproteins. In fungi, the enzyme plays a role in the assimilation of D-amino acids for cell growth and also in the detoxification of D-amino acid toxicity (2, 3). In vertebrates, this enzyme is mainly localized in the liver and kidney, and it metabolizes both exogenous and endogenous D-amino acids (2). In addition, the enzyme plays a specific role in the regulation of D-serine, a coagonist for the N-methyl-Daspartate receptor, in the brain (2).In contrast, for a long time, it was thought that DAO did not exist in prokaryotic organisms. However, this enzyme was recently identified in Arthrobacter protophormiae (ApDAO) and Streptomyces coelicolor (ScDAO) (4, 5). In addition to these bacterial species, a search of a prokaryotic genome database revealed a wide distribution of DAO homologous proteins in bacteria, especially a...

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Section: Resultssupporting

confidence: 69%

Section: Discussionmentioning

confidence: 88%

mentioning

confidence: 99%

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A Highly Stable d -Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus

Takahashi

Furukawara

Omae

et al. 2014

Appl Environ Microbiol

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“…The recombinant TPP Vmax was 290.0 ± 3.79 µmol.min -1 , and K M was 3.19 ± 0.10 mM, which is on the same order of magnitude of most of the reported TPPs: 2.5 ± 0.1 mM (Seo et al, 2000), 1.5 mM (Silva et al, 2005), 6.1 ± 1.5 mM (Nobre et al, 2008), 0.42 mM (Kushwaha et al, 2011), 0.39 ± 0.16 mM (Li et al, 2012), 0.36 ± 0.06 mM (Farelli et al, 2014), 0.87± 0.06 mM (Miao et al, 2016), 0.48 mM (Shan et al 2016), 0.23 ± 0.07, 0.50 ± 0.10, 0.69 ± 0.07 and 0.31 ± 0.04 mM (Liu et al, 2017a).…”

Section: Resultssupporting

confidence: 60%

Kinetic characterization and molecular modeling of trehalose-6-phosphate phosphatase from Anopheles gambiae and expressed in Pichia pastoris

Pessoa¹,

Izumi²,

Zanotto³

et al. 2017

Afr. J. Biotechnol.

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Trehalose-6-phosphate phosphatase (TPP) is one of the primary enzymes involved in the synthesis of trehalose, the main sugar found in insect hemolymph. In the present study, we report for the first time heterologous expression in Pichia pastoris, characterization and homology modeling of TPP from Anopheles gambiae mosquito. Purified TPP recombinant exhibited a molecular weight of approximately 36 kDa with optimum pH of 8.0, optimum temperature of 38°C, and the K M was 3.19 ± 0.10 mM. Inhibition tests revealed that CaCl 2 and Ca(NO 3 ) 2 at 5 and 25 mM, respectively, were effective inhibitors of TPP activity. Homology studies and molecular modeling indicated high similarity of the TPP tridimensional structure with TPP enzymes deposited at a data bank and these homology studies confirmed that it is a trehalose phosphatase with conserved motifs of the superfamily haloacid dehydrogenase. These results may provide support for the discovering of TPP inhibitors to be used for development of insecticides to control mosquito vectors.

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“…S4 in the supplemental material), with Acidithio-bacillus species displaying Fe 3ϩ sensitivity and Rubrobacter species having an affinity for ferric iron. Although high concentrations of ferric iron can lead to oxidative stress on microbes (71), several strategies, including the synthesis of trehalose (72) and radioresistance mechanisms (73)(74)(75), have been found in Rubrobacter species to resist oxidative stress. Second, patterns of Ferroplasma and Leptospirillum abundance were apparently shaped by tailing pH.…”

Section: Discussionmentioning

confidence: 99%

Correlating Microbial Diversity Patterns with Geochemistry in an Extreme and Heterogeneous Environment of Mine Tailings

Liu

Hua

Chen

et al. 2014

Appl Environ Microbiol

166

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Recent molecular surveys have advanced our understanding of the forces shaping the large-scale ecological distribution of microbes in Earth's extreme habitats, such as hot springs and acid mine drainage. However, few investigations have attempted dense spatial analyses of specific sites to resolve the local diversity of these extraordinary organisms and how communities are shaped by the harsh environmental conditions found there. We have applied a 16S rRNA gene-targeted 454 pyrosequencing approach to explore the phylogenetic differentiation among 90 microbial communities from a massive copper tailing impoundment generating acidic drainage and coupled these variations in community composition with geochemical parameters to reveal ecological interactions in this extreme environment. Our data showed that the overall microbial diversity estimates and relative abundances of most of the dominant lineages were significantly correlated with pH, with the simplest assemblages occurring under extremely acidic conditions and more diverse assemblages associated with neutral pHs. The consistent shifts in community composition along the pH gradient indicated that different taxa were involved in the different acidification stages of the mine tailings. Moreover, the effect of pH in shaping phylogenetic structure within specific lineages was also clearly evident, although the phylogenetic differentiations within the Alphaproteobacteria, Deltaproteobacteria, and Firmicutes were attributed to variations in ferric and ferrous iron concentrations. Application of the microbial assemblage prediction model further supported pH as the major factor driving community structure and demonstrated that several of the major lineages are readily predictable. Together, these results suggest that pH is primarily responsible for structuring whole communities in the extreme and heterogeneous mine tailings, although the diverse microbial taxa may respond differently to various environmental conditions.

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A Unique Combination of Genetic Systems for the Synthesis of Trehalose in Rubrobacter xylanophilus : Properties of a Rare Actinobacterial TreT

Cited by 35 publications

References 40 publications

A Highly Stable d -Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus

A Highly Stable d -Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus

Kinetic characterization and molecular modeling of trehalose-6-phosphate phosphatase from Anopheles gambiae and expressed in Pichia pastoris

Correlating Microbial Diversity Patterns with Geochemistry in an Extreme and Heterogeneous Environment of Mine Tailings

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