Structure-function analysis of two closely related cutinases from Thermobifida cellulosilytica

Jenny Arnling Bååth, Vera Novy, Leonor Vieira Carneiro, Georg M. Guebitz, Lisbeth Olsson, Peter Westh, Doris Ribitsch*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

Cutinases can play a significant role in a biotechnology-based circular economy. However, relatively little is known about the structure-function relationship of these enzymes, knowledge that is vital to advance optimized, engineered enzyme candidates. Here, two almost identical cutinases from Thermobifida cellulosilytica DSM44535 (ThcCut1 and ThcCut2) with only 18 amino acids difference were used for a rigorous biochemical characterization of their ability to hydrolyze PET, PET-model substrates, and cutin-model substrates. Kinetic parameters were compared with detailed in-silico docking studies of enzyme-ligand interactions. The two enzymes interacted with, and hydrolyzed PET differently, with ThcCut1 generating smaller PET-degradation products. ThcCut1 also showed higher catalytic efficiency on long-chain aliphatic substrates, an effect likely caused by small changes in the binding architecture. ThcCut2, in contrast, showed improved binding and catalytic efficiency when approaching the glass transition temperature of PET, an effect likely caused by longer amino acid residues in one area at the enzyme’s surface. Finally, the position of the single residue Q93 close to the active site, rotated out in ThcCut2, influenced the ligand position of a trimeric PET-model substrate. In conclusion, we illustrate that even minor sequence differences in cutinases can affect their substrate binding, substrate specificity, and catalytic efficiency drastically.
Original languageEnglish
JournalBiotechnology and Bioengineering
Volume119
Issue number2
Pages (from-to)470-481
Number of pages11
ISSN0006-3592
DOIs
Publication statusPublished - 2022

Keywords

  • Cutinase
  • Enzyme kinetics
  • PET hydrolase
  • Structure-function analysis
  • Substrate specificity

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