Plant Nitrilase Homologues in Fungi: Phylogenetic and Functional Analysis with Focus on Nitrilases in <i>Trametes versicolor</i> and <i>Agaricus bisporus</i>

• Main Authors: Lenka Rucká, Natalia Kulik, Petr Novotný, Anastasia Sedova, Lucie Petrásková, Romana Příhodová, Barbora Křístková, Petr Halada, Miroslav Pátek, Ludmila Martínková
• Format: Article
• Language: English
• Published: MDPI AG 2020-08-01
• Series: Molecules
• Subjects: fungi; plant nitrilase homologues; β-cyano-L-alanine; arylaliphatic nitriles; fumaronitrile; substrate specificity
• Online Access: https://www.mdpi.com/1420-3049/25/17/3861

Fungi contain many plant-nitrilase (NLase) homologues according to database searches. In this study, enzymes NitTv1 from <i>Trametes versicolor</i> and NitAb from <i>Agaricus bisporus</i> were purified and characterized as the representatives of this type of fungal NLase. Both enzymes were slightly more similar to NIT4 type than to NIT1/NIT2/NIT3 type of plant NLases in terms of their amino acid sequences. Expression of the synthetic genes in <i>Escherichia coli</i> Origami B (DE3) was induced with 0.02 mM isopropyl β-D-1-thiogalactopyranoside at 20 °C. Purification of NitTv1 and NitAb by cobalt affinity chromatography gave ca. 6.6 mg and 9.6 mg of protein per 100 mL of culture medium, respectively. Their activities were determined with 25 mM of nitriles in 50 mM Tris/HCl buffer, pH 8.0, at 30 °C. NitTv1 and NitAb transformed β-cyano-L-alanine (β-CA) with the highest specific activities (ca. 132 and 40 U mg⁻¹, respectively) similar to plant NLase NIT4. β-CA was transformed into Asn and Asp as in NIT4 but at lower Asn:Asp ratios. The fungal NLases also exhibited significant activities for (aryl)aliphatic nitriles such as 3-phenylpropionitrile, cinnamonitrile and fumaronitrile (substrates of NLase NIT1). NitTv1 was more stable than NitAb (at pH 5–9 vs. pH 5–7). These NLases may participate in plant–fungus interactions by detoxifying plant nitriles and/or producing plant hormones. Their homology models elucidated the molecular interactions with various nitriles in their active sites.