Phylogenetic analysis and in-depth characterization of functionally and structurally diverse CE5 cutinases

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, however, requires deeper knowledge of cutinases' biodiversity and structure-function relationships. Here, we mined over 3000 members from carbohydrate esterase family 5 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, which showed that cutinases with available crystal structures were phylogenetically closely related. We then selected nine phylogenic diverse cutinases for recombinant production and characterized their kinetic activity against para-nitrophenol substrates esterified with consecutively longer alkyl chains (pNP-C₂ to C₁₆). Each investigated cutinase had a unique activity fingerprint against the tested pNP substrates. The five enzymes with the highest activity on pNP-C₁₂ and C₁₆, 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, whereas 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 toward pNP-C₁₆. 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.