Relevance of aromatic and polar amino acids in the specificity of Inulinase ISO3 from Kluyveromyces marxianus: A molecular dynamics approach with an experimental verification

• Published in: International journal of biological macromolecules Vol. 242; no. Pt 1; p. 124734
• Main Authors: Trapala, Jonathan, González-Andrade, Martín, Olvera, Clarita, Cayetano-Cruz, Maribel, Sanz-Aparicio, Julia, Jimenez-Ortega, Elena, Bustos-Jaimes, Ismael, Montiel, Carmina
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2023
• Subjects: Amino Acids - metabolism; Fructans - metabolism; Glycoside Hydrolases - chemistry; Hydrolysis of agavin; Inulinase; Kluyveromyces - genetics; Molecular dynamics; Molecular Dynamics Simulation; Site-directed mutagenesis; Sucrose - metabolism; Molecular dynamics; Hydrolysis of agavin; Inulinase; Site-directed mutagenesis
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2023.124734

The Inulinase from Kluyveromyces marxianus ISO3 (Inu-ISO3) is an enzyme able to hydrolyze linear fructans such as chicory inulin as well as branched fructans like agavin. This enzyme was cloned and expressed in Komagataella pastoris to study the role of selected aromatic and polar residues in the catalytic pocket by Alanine scanning. Molecular dynamics (MD) simulations and enzyme kinetics analysis were performed to study the functional consequences of these amino acid substitutions. Site-directed mutagenesis was used to construct the mutants of the enzyme after carrying out the MD simulations between Inu-ISO3 and its substrates. Mutation Trp79:Ala resulted in the total loss of activity when fructans were used as substrates, while with sucrose, the activity decreased by 98 %. In contrast, the mutations Phe113:Ala and Gln236:Ala increased the invertase activity when sucrose was used as a substrate. Although these amino acids are not part of the conserved motifs where the catalytic triad is located, they are essential for the enzyme's activity. In silico and experimental approaches corroborate the relevance of these residues for substrate binding and their influence on enzymatic activity.