Novy V

References (2)

Title : Structure-function analysis of two closely related cutinases from Thermobifida cellulosilytica - Baath_2021_Biotechnol.Bioeng__
Author(s) : Baath JA , Novy V , Carneiro LV , Guebitz GM , Olsson L , Westh P , Ribitsch D
Ref : Biotechnol Bioeng , : , 2021
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 (Thc_Cut1 and Thc_Cut2) 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 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. This article is protected by copyright. All rights reserved.
ESTHER : Baath_2021_Biotechnol.Bioeng__
PubMedSearch : Baath_2021_Biotechnol.Bioeng__
PubMedID: 34755331
Gene_locus related to this paper: thefu-q6a0i4 , thefu-q6a0i3

Title : Phylogenetic analysis and in-depth characterization of functionally and structurally diverse CE5 cutinases - Novy_2021_J.Biol.Chem__101302
Author(s) : Novy V , Carneiro LV , Shin JH , Larsbrink J , Olsson L
Ref : Journal of Biological Chemistry , :101302 , 2021
Abstract : Cutinases are esterases that release fatty acids from the apoplastic layer in plants. As they accept bulky and hydrophobic substrates, cutinases could be used in many applications, ranging from valorization of bark-rich side streams to plastic recycling. Advancement of these applications with cutinases as biocatalysts, however, requires deeper knowledge of the enzymes' biodiversity and structure-function relationships. Here, we mined over 3000 members from Carbohydrate Esterase family 5 (CE5) for putative cutinases and condensed it to 151 genes from known or putative lignocellulose-targeting organisms. The 151 genes were subjected to a phylogenetic analysis. While cutinases with available crystal structures were phylogenetically closely related, we selected nine phylogenic diverse cutinases for characterization. The nine selected cutinases were recombinantly produced and their kinetic activity was characterized against para-nitrophenol substrates esterified with consecutively longer alkyl chains (pNP-C(2) to C(16)). The investigated cutinases each had a unique activity fingerprint against tested pNP-substrates. The five enzymes with the highest activity on pNP-C(12) and C(16), indicative of activity on bulky hydrophobic compounds, were selected for in-depth kinetic and structure-function analysis. All five enzymes showed a decrease in k(cat) values with increasing substrate chain length, while K(M) values and binding energies (calculated from in silico docking analysis) improved. Two cutinases from Fusarium solani and Cryptococcus sp. exhibited outstandingly low K(M) values, resulting in high catalytic efficiencies towards pNP-C(16). Docking analysis suggested that different clades of the phylogenetic tree may harbor enzymes with different modes of substrate interaction, involving a solvent-exposed catalytic triad, a lipase-like lid, or a clamshell-like active site possibly formed by flexible loops.
ESTHER : Novy_2021_J.Biol.Chem__101302
PubMedSearch : Novy_2021_J.Biol.Chem__101302
PubMedID: 34653507
Gene_locus related to this paper: 9pezi-s4vch4 , aspfu-q4x1n0 , crysp-Q874E9 , emeni-q5b2c1 , fusso-cutas , pyrbr-Q9Y7G8 , copci-b9u443 , thite-g2rae6 , strsw-c9zcr8