Considerations Regarding Activity Determinants of Fungal Polyphenol Oxidases Based on Mutational and Structural Studies.

Autor: Nikolaivits E; Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, Greece., Valmas A; Department of Biology, University of Patras, Patras, Greece., Dedes G; Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, Greece., Topakas E; Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, Greece., Dimarogona M; Laboratory of Structural Biology and Biotechnology, Department of Chemical Engineering, University of Patras, Patras, Greece mdimarog@chemeng.upatras.gr.
Jazyk: angličtina
Zdroj: Applied and environmental microbiology [Appl Environ Microbiol] 2021 May 11; Vol. 87 (11). Date of Electronic Publication: 2021 May 11 (Print Publication: 2021).
DOI: 10.1128/AEM.00396-21
Abstrakt: Polyphenol oxidases (PPOs) are an industrially relevant family of enzymes, being involved in the postharvest browning of fruits and vegetables, as well as in human melanogenesis. Their involvement lies in their ability to oxidize phenolic or polyphenolic compounds, which subsequently form pigments. The 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 better understand the function of a previously characterized PPO from Thermothelomyces thermophila ( Tt PPO). The crystal structure of a Tt PPO variant, determined at 1.55 Å resolution, represents the second known structure of an ascomycete PPO. Kinetic data for structure-guided mutants prove the implication of "gate" residue L306, residue H B1 +1 (G292), and H B2 +1 (Y296) in Tt PPO function against various substrates. Our findings demonstrate the role of L306 in the accommodation of bulky substrates and show that residue H B1 +1 is unlikely to determine monophenolase activity, as was suggested from previous studies. IMPORTANCE PPOs are enzymes of biotechnological interest. They have been extensively studied both biochemically and structurally, with a special focus on the plant-derived counterparts. Even so, explicit description of the molecular determinants of their substrate specificity is still pending. For ascomycete PPOs, only one crystal structure has been determined so far, thus limiting our knowledge on this tree branch of the family. In the present study, we report the second crystal structure of an ascomycete PPO. Combined with site-directed mutagenesis and biochemical studies, we depict the amino acids in the vicinity of the active site that affect enzyme activity and perform a detailed analysis on a variety of substrates. Our findings improve current understanding of structure-function relations of microbial PPOs, which is a prerequisite for the engineering of biocatalysts of desired properties.
(Copyright © 2021 American Society for Microbiology.)
Databáze: MEDLINE
Externí odkaz: