Identification of the amino acid position controlling the different enzymatic activities in walnut tyrosinase isoenzymes (jrPPO1 and jrPPO2)

Panis, Felix; Rompel, Annette

doi:10.1038/s41598-020-67415-6

Cited by 15 publications

(18 citation statements)

References 55 publications

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“…However, samples from the V treatment did not react with protocatechuic acid or L-tyrosine, which indicated a low level of PPO activity. Compared with JrPPO2, JrPPO1 exerts greater activity towards monophenols and does not accept protocatechuic acid as substrate, whereas JrPPO2 is more active towards o -diphenols [ 30 ]. Therefore, our results suggest that there is high JrPPO2 activity under treatment A.…”

Section: Resultsmentioning

confidence: 99%

“…To determine whether JrPPO1 or JrPPO2 exerted high activity in different stages or tissues, a simple browning assay was performed essentially as described by Panis [ 30 ]. Total protein (via a supernatant) was isolated from equal weights of explants or different tissues as described above.…”

Section: Methodsmentioning

confidence: 99%

“…A second homolog of PPO ( JrPPO2 ) was discovered on the basis of the J. regia genome sequence, but its mechanism of action is substantially less clear [ 29 ]. Panis reported that, compared with JrPPO2, JrPPO1 exhibited a greater activity towards monophenols, whereas JrPPO2 was more active towards o -diphenols [ 30 ].…”

Section: Introductionmentioning

confidence: 99%

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The role of JrPPOs in the browning of walnut explants

Zhao

Wang²,

Li³

et al. 2021

BMC Plant Biol

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Background Tissue culture is an effective method for the rapid breeding of seedlings and improving production efficiency, but explant browning is a key limiting factor of walnut tissue culture. Specifically, the polymerization of PPO-derived quinones that cause explant browning of walnut is not well understood. This study investigated explants of ‘Zanmei’ walnut shoot apices cultured in agar (A) or vermiculite (V) media, and the survival percentage, changes in phenolic content, POD and PPO activity, and JrPPO expression in explants were studied to determine the role of PPO in the browning of walnut explants. Results The results showed that the V media greatly reduced the death rate of explants, and 89.9 and 38.7% of the explants cultured in V media and A media survived, respectively. Compared with that of explants at 0 h, the PPO of explants cultured in A was highly active throughout the culture, but activity in those cultured in V remained low. The phenolic level of explants cultured in A increased significantly at 72 h but subsequently declined, and the content in the explants cultured in V increased to a high level only at 144 h. The POD in explants cultured in V showed high activity that did not cause browning. Gene expression assays showed that the expression of JrPPO1 was downregulated in explants cultured in both A and V. However, the expression of JrPPO2 was upregulated in explants cultured in A throughout the culture and upregulated in V at 144 h. JrPPO expression analyses in different tissues showed that JrPPO1 was highly expressed in stems, young leaves, mature leaves, catkins, pistils, and hulls, and JrPPO2 was highly expressed in mature leaves and pistils. Moreover, browning assays showed that both explants in A and leaf tissue exhibited high JrPPO2 activity. Conclusion The rapid increase in phenolic content caused the browning and death of explants. V media delayed the rapid accumulation of phenolic compounds in walnut explants in the short term, which significantly decreased explants mortality. The results suggest that JrPPO2 plays a key role in the oxidation of phenols in explants after branch injury.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The role of JrPPOs in the browning of walnut explants

Zhao

Wang²,

Li³

et al. 2021

BMC Plant Biol

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“…In fact, a mechanism for the hydroxylation by Ao CO4 has been proposed, where the enzyme’s copper site is in hydroperoxide -form and monooxygenation occurs by electrophilic aromatic substitution without the need for deprotonation (5). Recently, docking studies revealed that the preference towards diphenolic substrates, exhibited by PPOs bearing a Gly in position H B1 +1, is explained by a “laying down” orientation of the substrate, which is stereochemically prohibited in PPOs bearing a bulkier Asn residue (15).…”

Section: Introductionmentioning

confidence: 99%

Considerations on activity determinants of fungal polyphenol oxidases based on mutational and structural studies

Nikolaivits

Valmas

Dedes

et al. 2021

Preprint

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Polyphenol oxidases (PPOs) are an industrially relevant family of enzymes, being involved in the post-harvest browning of fruits and vegetables, as well as in human melanogenesis. Their involvement lies in their ability to oxidize phenolic or polyphenolic compounds, that subsequently form pigments. PPO family includes tyrosinases and catechol oxidases, which in spite of their high structural similarity, exhibit different catalytic activities. Long-standing research efforts have not yet managed to decipher the structural determinants responsible for this differentiation, as every new theory is disproved by a more recent study. In the present work, we combined biochemical along with structural data, in order to rationalize the function of a previously characterized PPO from Thermothelomyces thermophila (TtPPO). The crystal structure of a TtPPO variant, determined at 1.55 Å resolution, represents the second known structure of an ascomycete PPO. Kinetic data of structure-guided mutants prove the implication of 'gate' residue L306, residue HB1+1 (G292) and HB2+1 (Y296) in TtPPO function against various substrates. Our findings demonstrate the role of L306 in the accommodation of bulky substrates and that residue HB1+1 is unlikely to determine monophenolase activity as suggested from previous studies.

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“…In spite of various crystallographic and molecular docking studies, the structural determinants that affect the distinction between the two enzymatic activities and the substrate specificity exhibited by PPOs are still not fully understood. For instance, molecular studies on two walnut (Juglans regia) tyrosinase isoenzymes (JrPPO1 and JrPPO2) showed that the activity towards monophenolic substrates was dependent on the occurrence of an asparagine residue located after one of the CuB coordinating histidines [11]. However, there are many PPOs that do not have an Asn residue in this position but still display monophenolase activity [7].…”

Section: Introductionmentioning

confidence: 99%

Structural Studies of a Fungal Polyphenol Oxidase with Application to Bioremediation of Contaminated Water

Valmas

Dedes

Dimarogona

2020

The 1st International Electronic Conference on Microbiology

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Polyphenol oxidases (PPOs) are a group of Cu-containing enzymes exhibiting two activities: catechol oxidase and tyrosinase. Their precise mechanism of action and the structural elements that determine the distinction between the two activities are yet to be fully understood. In nature, PPOs catalyze the oxidation of several phenols to o-quinones, considerably affecting the color and nutritional properties of numerous agricultural products. On the other hand, PPOs have been widely employed as biocatalysts in food, pharmaceutical, and cosmetic industries. TtPPO is a PPO from the thermophilic fungus Thermothelomyces thermophila (TtPPO), capable of degrading of chlorophenols (CPs), contagious by-products of various pesticides. The present work aims to clarify the structural determinants of TtPPO function, by performing protein-ligand docking experiments via YASARA software. The docking results are compared with biochemical data, and the role of specific amino acids in TtPPO function is investigated. The identification of the amino acids involved in binding of the different substrates to the active site of the enzyme would allow the structure-based design of a more efficient biocatalyst for wastewater treatment.

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Identification of the amino acid position controlling the different enzymatic activities in walnut tyrosinase isoenzymes (jrPPO1 and jrPPO2)

Cited by 15 publications

References 55 publications

The role of JrPPOs in the browning of walnut explants

The role of JrPPOs in the browning of walnut explants

Considerations on activity determinants of fungal polyphenol oxidases based on mutational and structural studies

Structural Studies of a Fungal Polyphenol Oxidase with Application to Bioremediation of Contaminated Water

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