The multicopper oxidase from the archaeon <i>Pyrobaculum aerophilum</i> shows nitrous oxide reductase activity

Fernandes, Ana; Damas, João M.; Todorović, Smilja; Huber, Robert; Baratto, Maria Camilla; Pogni, Rebecca; Soares, Cláudio M.; Martins, Lı́gia O.

doi:10.1111/j.1742-4658.2010.07725.x

Cited by 51 publications

(49 citation statements)

References 72 publications

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“…Same may be said for other bacterial laccases. For example, the spore coat protein CotA from B. subtilis is a model bacterial laccase, yet it has been shown to be a billirubin oxidase (Sakasegawa et al 2006) and can also use N 2 O as the electron acceptor (Fernandes et al 2010), which is not typical for a laccase. Similarly, CueO from E. coli can oxidize many of the classical laccase substrates, but it has been shown to have a strong cuprous oxidase activity and is, in fact, involved in vivo in the copper homeostasis (Singh et al 2004).…”

Section: Discussionmentioning

confidence: 98%

“…Among these, only the multicopper oxidase McoP from the archaeon Pyrobaculum aerophilum has been studied previously at the protein level. It has been shown to use not only dioxygen but also nitrous oxide as an electron acceptor and could thus play a role in denitrification (Fernandes et al 2010). Multiple sequence alignment of ThioLacc with CueO and McoP as well as the best-studied laccase CotA from Bacillus subtilis is shown in Fig.…”

Section: Sequence Analysis Of Thiolaccmentioning

confidence: 99%

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Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp

Ausec

Črnigoj

Šnajder

et al. 2015

Appl Microbiol Biotechnol

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Laccases are oxidoreductases mostly studied in fungi, while bacterial laccases remain poorly studied despite their high genetic diversity and potential for biotechnological application. Our previous bioinformatic analysis identified alkaliphilic bacterial strains Thioalkalivibrio sp. as potential sources of robust bacterial laccases that would be stable at high pH. In the present work, a gene for a laccase-like enzyme from Thioalkalivibrio sp. ALRh was cloned and expressed as a 6× His-tagged protein in Escherichia coli. The purified enzyme was a pH-tolerant laccase stable in the pH range between 2.1 and 9.9 at 20 °C as shown by intrinsic fluorescence emission spectrometry. It had optimal activities at pH 5.0 and pH 9.5 with the laccase substrates 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol, respectively. In addition, it could oxidize several other monophenolic compounds and potassium hexacyanoferrate(II) but not tyrosine. It showed highest activity at 50 °C, making it suitable for prolonged incubations at this temperature. The present study shows that Thioalkalivibrio sp. encodes an active, alkaliphilic, and thermo-tolerant laccase and contributes to our understanding of the versatility of bacterial laccase-like multicopper oxidases in general.

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Section: Discussionmentioning

confidence: 98%

Section: Sequence Analysis Of Thiolaccmentioning

confidence: 99%

Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp

Ausec

Črnigoj

Šnajder

et al. 2015

Appl Microbiol Biotechnol

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“…The laccase has later been shown to oxidize a large variety of substrates, such as substituted aromatic compounds [16] and a number of important dyes [17]. In addition, it can function as a bilirubin oxidase [18], a low-affinity nitrous oxide reductase [19], and a manganese oxidase [20].…”

Section: Introductionmentioning

confidence: 99%

Characterization of an Alkali- and Halide-Resistant Laccase Expressed in E. coli: CotA from Bacillus clausii

2014

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The limitations of fungal laccases at higher pH and salt concentrations have intensified the search for new extremophilic bacterial laccases. We report the cloning, expression, and characterization of the bacterial cotA from Bacillus clausii, a supposed alkalophilic ortholog of cotA from B. subtilis. Both laccases were expressed in E. coli strain BL21(DE3) and characterized fully in parallel for strict benchmarking. We report activity on ABTS, SGZ, DMP, caffeic acid, promazine, phenyl hydrazine, tannic acid, and bilirubin at variable pH. Whereas ABTS, promazine, and phenyl hydrazine activities vs. pH were similar, the activity of B. clausii cotA was shifted upwards by ∼0.5–2 pH units for the simple phenolic substrates DMP, SGZ, and caffeic acid. This shift is not due to substrate affinity (KM) but to pH dependence of catalytic turnover: The kcat of B. clausii cotA was 1 s−1 at pH 6 and 5 s−1 at pH 8 in contrast to 6 s−1 at pH 6 and 2 s−1 at pH 8 for of B. subtilis cotA. Overall, kcat/KM was 10-fold higher for B. subtilis cotA at pHopt. While both proteins were heat activated, activation increased with pH and was larger in cotA from B. clausii. NaCl inhibited activity at acidic pH, but not up to 500–700 mM NaCl in alkaline pH, a further advantage of the alkali regime in laccase applications. The B. clausii cotA had ∼20 minutes half-life at 80°C, less than the ∼50 minutes at 80°C for cotA from B. subtilis. While cotA from B. subtilis had optimal stability at pH∼8, the cotA from B. clausii displayed higher combined salt- and alkali-resistance. This resistance is possibly caused by two substitutions (S427Q and V110E) that could repel anions to reduce anion-copper interactions at the expense of catalytic proficiency, a trade-off of potential relevance to laccase optimization.

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“…In several denitrificant microorganisms, N 2 O is finally reduced to N 2 by a periplasmic reductase (NosZ). However, N 2 may also be produced in certain microorganisms lacking NosZ homologues, supporting the possibility that a different kind of N 2 O reductase might exist (5).…”

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confidence: 99%

Parallel Pathways for Nitrite Reduction during Anaerobic Growth in Thermus thermophilus

et al. 2014

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Respiratory reduction of nitrate and nitrite is encoded in Thermus thermophilus by the respective transferable gene clusters. Nitrate is reduced by a heterotetrameric nitrate reductase (Nar) encoded along transporters and regulatory signal transduction systems within the nitrate respiration conjugative element (NCE). The nitrite respiration cluster (nic) encodes homologues of nitrite reductase (Nir) and nitric oxide reductase (Nor). The expression and role of the nirSJM genes in nitrite respiration were analyzed. The three genes are expressed from two promoters, one (nirSp) producing a tricistronic mRNA under aerobic and anaerobic conditions and the other (nirJp) producing a bicistronic mRNA only under conditions of anoxia plus a nitrogen oxide. As for its nitrite reductase homologues, NirS is expressed in the periplasm, has a covalently bound heme c, and conserves the heme d 1 binding pocket. NirJ is a cytoplasmic protein likely required for heme d 1 synthesis and NirS maturation. NirM is a soluble periplasmic homologue of cytochrome c 552 . Mutants defective in nirS show normal anaerobic growth with nitrite and nitrate, supporting the existence of an alternative Nir in the cells. Gene knockout analysis of different candidate genes did not allow us to identify this alternative Nir protein but revealed the requirement for Nar in NirS-dependent and NirS-independent nitrite reduction. As the likely role for Nar in the process is in electron transport through its additional cytochrome c periplasmic subunit (NarC), we concluded all the Nir activity takes place in the periplasm by parallel pathways.

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The multicopper oxidase from the archaeon Pyrobaculum aerophilum shows nitrous oxide reductase activity

Cited by 51 publications

References 72 publications

Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp

Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp

Characterization of an Alkali- and Halide-Resistant Laccase Expressed in E. coli: CotA from Bacillus clausii

Parallel Pathways for Nitrite Reduction during Anaerobic Growth in Thermus thermophilus

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