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In crystallo activity tests with latent apple tyrosinase and two mutants reveal the importance of the mutated sites for polyphenol oxidase activity

In crystallo activity tests with latent apple tyrosinase and two mutants reveal the importance of... Tyrosinases are type 3 copper enzymes that belong to the polyphenol oxidase (PPO) family and are able to catalyze both the ortho‐hydroxylation of monophenols and their subsequent oxidation to o‐quinones, which are precursors for the biosynthesis of colouring substances such as melanin. The first plant pro‐tyrosinase from Malus domestica (MdPPO1) was recombinantly expressed in its latent form (56.4 kDa) and mutated at four positions around the catalytic pocket which are believed to influence the activity of the enzyme. Mutating the amino acids, which are known as activity controllers, yielded the mutants MdPPO1‐Ala239Thr and MdPPO1‐Leu243Arg, whereas mutation of the so‐called water‐keeper and gatekeeper residues resulted in the mutants MdPPO1‐Glu234Ala and MdPPO1‐Phe259Ala, respectively. The wild‐type enzyme and two of the mutants, MdPPO1‐Ala239Thr and MdPPO1‐Phe259Ala, were successfully crystallized, leading to single crystals that diffracted to 1.35, 1.55 and 1.70 Å resolution, respectively. All crystals belonged to space group P212121, exhibiting similar unit‐cell parameters: a = 50.70, b = 80.15, c = 115.96 Å for the wild type, a = 50.58, b = 79.90, c = 115.76 Å for MdPPO1‐Ala239Thr and a = 50.53, b = 79.76, c = 116.07 Å for MdPPO1‐Phe259Ala. In crystallo activity tests with the crystals of the wild type and the two mutants were performed by adding the monophenolic substrate tyramine and the diphenolic substrate dopamine to crystal‐containing drops. The effects of the mutation on the activity of the enzyme were observed by colour changes of the crystals owing to the conversion of the substrates to dark chromophore products. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Crystallographica Section F Wiley

In crystallo activity tests with latent apple tyrosinase and two mutants reveal the importance of the mutated sites for polyphenol oxidase activity

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Publisher
Wiley
Copyright
Copyright © 2017 Wiley Subscription Services
ISSN
2053-230X
eISSN
2053-230X
DOI
10.1107/S2053230X17010822
pmid
28777094
Publisher site
See Article on Publisher Site

Abstract

Tyrosinases are type 3 copper enzymes that belong to the polyphenol oxidase (PPO) family and are able to catalyze both the ortho‐hydroxylation of monophenols and their subsequent oxidation to o‐quinones, which are precursors for the biosynthesis of colouring substances such as melanin. The first plant pro‐tyrosinase from Malus domestica (MdPPO1) was recombinantly expressed in its latent form (56.4 kDa) and mutated at four positions around the catalytic pocket which are believed to influence the activity of the enzyme. Mutating the amino acids, which are known as activity controllers, yielded the mutants MdPPO1‐Ala239Thr and MdPPO1‐Leu243Arg, whereas mutation of the so‐called water‐keeper and gatekeeper residues resulted in the mutants MdPPO1‐Glu234Ala and MdPPO1‐Phe259Ala, respectively. The wild‐type enzyme and two of the mutants, MdPPO1‐Ala239Thr and MdPPO1‐Phe259Ala, were successfully crystallized, leading to single crystals that diffracted to 1.35, 1.55 and 1.70 Å resolution, respectively. All crystals belonged to space group P212121, exhibiting similar unit‐cell parameters: a = 50.70, b = 80.15, c = 115.96 Å for the wild type, a = 50.58, b = 79.90, c = 115.76 Å for MdPPO1‐Ala239Thr and a = 50.53, b = 79.76, c = 116.07 Å for MdPPO1‐Phe259Ala. In crystallo activity tests with the crystals of the wild type and the two mutants were performed by adding the monophenolic substrate tyramine and the diphenolic substrate dopamine to crystal‐containing drops. The effects of the mutation on the activity of the enzyme were observed by colour changes of the crystals owing to the conversion of the substrates to dark chromophore products.

Journal

Acta Crystallographica Section FWiley

Published: Jan 1, 2017

Keywords: ; ; ; ;

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