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/lp/wiley/in-crystallo-activity-tests-with-latent-apple-tyrosinase-and-two-mF0ud58kBA
- 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 F – Wiley
Published: Jan 1, 2017
Keywords: ; ; ; ;