Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr215 in Aerococcus viridans lactate oxidase

Stoisser, Thomas; Brunsteiner, Michael; Wilson, D. Keith; Nidetzky, Bernd

doi:10.1038/srep27892

Cited by 39 publications

(48 citation statements)

References 54 publications

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“…As a control we used in-house produced AvLCTO with both affinity tag (tag cleaved later during purification) and tag free version. Both of these versions of AvLCTO had enzymatic activity similar to earlier reports [22]. We also tested other substrates such as glycolate and 4-hydroxy mandelate with PaLCTO but no activity could be seen.…”

Section: Discussionsupporting

confidence: 76%

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FMN-dependent oligomerization of putative lactate oxidase fromPediococcus acidilactici

Ashok

Maksimainen

Kallio

et al. 2019

Preprint

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13Lactate oxidases belong to a group of FMN-dependent enzymes and they catalyze a conversion 14 of lactate to pyruvate with a release of hydrogen peroxide. Hydrogen peroxide is also utilized 15 as a read out in biosensors to quantitate lactate levels in biological samples. Aerococcus 16 viridans lactate oxidase is the best characterized lactate oxidase and our knowledge of lactate 17 oxidases relies largely to studies conducted with that particular enzyme. Pediococcus 18 acidilactici lactate oxidase is also commercially available for e.g. lactate measurements, but 19 this enzyme has not been characterized before in detail. Here we report structural 20 characterization of the recombinant enzyme and its co-factor dependent oligomerization. The 21 crystal structures revealed two distinct conformations in the loop closing the active site, 22 consistent with previous biochemical studies implicating the role of loop in catalysis. Despite 23 the structural conservation of active site residues when compared to Aerococcus viridans 24 lactate oxidase we were not able to detect either oxidase or monooxygenase activity when L-25 lactate or other potential alpha hydroxyl acids were used as a substrate. Pediococcus 26 acidilactici lactate oxidase is therefore an example of a misannotation of an FMN-dependent 27 enzyme, which catalyzes likely a so far unknown oxidation reaction. 28 3 29 4 53 Pediococcus acidilactici proteome does not use lactate as a substrate. We describe here 54 structural and biophysical studies of PaLCTO, which revealed FMN-dependent folding and 55 oligomerization of the enzyme. 56 57 Materials and methods 58 Cloning and site directed mutagenesis 59 Pediococcus acidilactici proteome wide search for keywords lactate, lactate oxidase, α-60 hydroxy acid in Uniprot database gave only one hit that matched with AvLCTO. Similarly, 61 BLASTp search with AvLCTO against P. acidilactici DSM20284 revealed the same hit 62 annotated as putative L-lactate oxidase (Uniprot E0NE46). Genomic DNA of Pediococcus 63 acidilactici (DSM 20284) and Aerococcus viridans (DSM 20340) was obtained from DSMZ, 64 Germany. Gene encoding PaLCTO was cloned into pNH-TrxT (Structural Genomics 65 Consortium) vector using SLIC cloning. The vector encodes for an N-terminal thioredoxin tag 66 with a cleavable TEV protease recognition site. A94G mutant was obtained using standard site-67 directed mutagenesis protocol. All clones were verified using Sanger's dideoxy sequencing. 68 Untagged PaLCTO and AvLCTO were recombinantly expressed from pNIC-CH vector with a 69 stop codon added immediately after the native sequence. Commercial PaLCTO was purchased 70 from Sigma (catalog number LO638, lots STBG2905V and STBF3223V).71 Protein expression and purification 72 Proteins were expressed using BL21 (DE3). Overnight culture was inoculated into terrific broth 73 auto-induction media supplemented with 8 g/L glycerol and 50 μg/ml kanamycin. Cells were 74 grown at 37°C until OD 600 reached 1. The temperature was reduced to 18°C for overnight for 5 75 protein e...

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Section: Discussionsupporting

confidence: 76%

“…Recent experimental evidence has shown that Tyr215 controls entry and exit of substrate and product in AvLCTO as mutations in Tyr215 result in a change in time of product release [22]. The corresponding residue in PaLCTO is Tyr214 and due to similarity, the loop could have a similar role in this enzyme.…”

Section: Resultsmentioning

confidence: 99%

FMN-dependent oligomerization of putative lactate oxidase fromPediococcus acidilactici

Ashok

Maksimainen

Kallio

et al. 2019

Preprint

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“…Protein structure analysis indicated that the replacement of Asp501 caused a decrease in the number of hydrogen bonds formed at position 501 and a larger space between Gly501 and Met 503 ( Figure S3), which may increase the conformational flexibility of this region. Conformational flexibility in the active site is important for substrate binding and for enzyme catalysis, and there is usually a positive correlation between conformational flexibility and enzyme activity [24][25][26]. As mentioned above, the Asp501 is adjacent to the axial ligand (Met502) of the T1 copper ion and lies at the surface of the water channel.…”

Section: Indigo Carmine Decolorizationmentioning

confidence: 99%

Improving the Indigo Carmine Decolorization Ability of a Bacillus amyloliquefaciens Laccase by Site-Directed Mutagenesis

Wang

Feng

2017

Catalysts

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Indigo carmine is a typical recalcitrant dye which is widely used in textile dyeing processes. Laccases are versatile oxidases showing strong ability to eliminate hazardous dyes from wastewater. However, most laccases require the participation of mediators for efficient decolorization of indigo carmine. Here we describe the improvement of the decolorization ability of a bacterial laccase through site-directed mutagenesis. A D501G variant of Bacillus amyloliquefaciens laccase was constructed and overexpressed in Escherichia coli. The laccase activity in the culture supernatant achieved 3374 U·L −1 for the mutant. Compared with the wild-type enzyme, the D501G exhibited better stability and catalytic efficiency. It could decolorize more than 92% of indigo carmine without additional mediators in 5 h at pH 9.0, which was 3.5 times higher than the wild-type laccase. Isatin sulfonic acid was confirmed to be the main product of indigo carmine degradation by UV-vis and LC-MS analyses.

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“…This is achieved by conformational dynamics of the loop. Substitutions in this residue were shown to reduce the rate of product release from the enzyme [12].…”

Section: Introductionmentioning

confidence: 99%

FMN-dependent oligomerization of putative lactate oxidase from Pediococcus acidilactici

et al. 2020

View full text Add to dashboard Cite

Lactate oxidases belong to a group of FMN-dependent enzymes and they catalyze a conversion of lactate to pyruvate with a release of hydrogen peroxide. Hydrogen peroxide is also utilized as a read out in biosensors to quantitate lactate levels in biological samples. Aerococcus viridans lactate oxidase is the best characterized lactate oxidase and our knowledge of lactate oxidases relies largely to studies conducted with that particular enzyme. Pediococcus acidilactici lactate oxidase is also commercially available for e.g. lactate measurements, but this enzyme has not been characterized in detail before. Here we report structural characterization of the recombinant enzyme and its co-factor dependent oligomerization. The crystal structures revealed two distinct conformations in the loop closing the active site, consistent with previous biochemical studies implicating the role of loop in catalysis. Despite the structural conservation of active site residues, we were not able to detect either oxidase or monooxygenase activity when L-lactate was used as a substrate. Pediococcus acidilactici lactate oxidase is therefore an example of a misannotation of an FMNdependent enzyme, which catalyzes likely a so far unknown oxidation reaction.

show abstract

Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr215 in Aerococcus viridans lactate oxidase

Cited by 39 publications

References 54 publications

FMN-dependent oligomerization of putative lactate oxidase fromPediococcus acidilactici

FMN-dependent oligomerization of putative lactate oxidase fromPediococcus acidilactici

Improving the Indigo Carmine Decolorization Ability of a Bacillus amyloliquefaciens Laccase by Site-Directed Mutagenesis

FMN-dependent oligomerization of putative lactate oxidase from Pediococcus acidilactici

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