Structure-function analysis of two closely related cutinases from Thermobifida cellulosilytica
Artikel i vetenskaplig tidskrift, 2021

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 (Thc_Cut1 and Thc_Cut2) with only 18 amino acids difference were used for a rigorous biochemical characterization of their ability to hydrolyze poly(ethylene terephthalate) (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 Thc_Cut1 generating smaller PET-degradation products. Thc_Cut1 also showed higher catalytic efficiency on long-chain aliphatic substrates, an effect likely caused by small changes in the binding architecture. Thc_Cut2, 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 Thc_Cut2, 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.

cutinase

PET hydrolase

structure-function analysis

enzyme kinetics

substrate specificity

Författare

Jenny Arnling Bååth

Danmarks Tekniske Universitet (DTU)

Vera Novy

Chalmers, Biologi och bioteknik, Industriell bioteknik

Leonor Vieira Carneiro

Chalmers, Biologi och bioteknik

Georg M. Guebitz

Universität für Bodenkultur

Lisbeth Olsson

Chalmers, Biologi och bioteknik, Industriell bioteknik

Peter Westh

Danmarks Tekniske Universitet (DTU)

Doris Ribitsch

Universität für Bodenkultur

Biotechnology and Bioengineering

0006-3592 (ISSN) 1097-0290 (eISSN)

Vol. In Press

Ämneskategorier

Biokemi och molekylärbiologi

Strukturbiologi

Biokatalys och enzymteknik

DOI

10.1002/bit.27984

PubMed

34755331

Mer information

Senast uppdaterat

2021-11-23