Fungi contain many plant-nitrilase (NLase) homologues according to database searches. In this study, enzymes NitTv1 from Trametes versicolor and NitAb from Agaricus bisporus 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 Escherichia coli 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.

Centre for Nanobiology and Structural Biology Institute of Microbiology Czech Academy of Sciences Zamek 136 CZ 373 33 Nové Hrady Czech Republic

Faculty of Biomedical Engineering Czech Technical University Prague nam Sítná 3105 CZ 272 01 Kladno Czech Republic

Laboratory of Biotransformation Institute of Microbiology Czech Academy of Sciences Vídeňská 1083 CZ 142 20 Prague Czech Republic

Laboratory of Modulation of Gene Expression Institute of Microbiology Czech Academy of Sciences Vídeňská 1083 CZ 142 20 Prague Czech Republic

Laboratory of Structural Biology and Cell Signaling BioCeV Institute of Microbiology Czech Academy of Sciences Průmyslová 595 CZ 252 50 Vestec Czech Republic

Zobrazit více v PubMed

Pace H.C., Brenner C. The nitrilase superfamily: Classification, structure and function. Genome Biol. 2001;2:REVIEWS0001. doi: 10.1186/gb-2001-2-1-reviews0001. PubMed DOI PMC

Brenner C. Catalysis in the nitrilase superfamily. Curr. Opin. Struct. Biol. 2002;12:775–782. doi: 10.1016/S0959-440X(02)00387-1. PubMed DOI

Thuku R.N., Brady D., Benedik M.J., Sewell B.T. Microbial nitrilases: Versatile, spiral forming, industrial enzymes. J. Appl. Microbiol. 2009;106:703–727. doi: 10.1111/j.1365-2672.2008.03941.x. PubMed DOI

Piotrowski M., Schönfelder S., Weiler E.W. The Arabidopsis thaliana isogene NIT4 and its orthologs in tobacco encode β-cyano-L-alanine hydratase/nitrilase. J. Biol. Chem. 2001;276:2616–2621. doi: 10.1074/jbc.M007890200. PubMed DOI

Martínková L., Rucká L., Nešvera J., Pátek M. Recent advances and challenges in the heterologous production of microbial nitrilases for biocatalytic applications. World J. Microbiol. Biotechnol. 2017;33:8. doi: 10.1007/s11274-016-2173-6. PubMed DOI

Prozomix. [(accessed on 14 August 2020)]; Available online: http://www.prozomix.com/products/view?product=1831.

Codexis 3. [(accessed on 14 August 2020)]; Available online: https://www.codexis-estore.com/product-page/codex-nitrilase-nit-screening-kit.

Merck KGaA. [(accessed on 14 August 2020)]; Available online: https://www.sigmaaldrich.com/catalog/product/sigma/04529.

Vorwerk S., Biernacki S., Hillebrand H., Janzik I., Müller A., Weiler E.W., Piotrowski M. Enzymatic characterization of the recombinant Arabidopsis thaliana nitrilase subfamily encoded by the NIT2/NIT1/NIT3-gene cluster. Planta. 2001;212:508–516. doi: 10.1007/s004250000420. PubMed DOI

Osswald S., Wajant H., Effenberger F. Characterization and synthetic applications of recombinant AtNIT1 from Arabidopsis thaliana. Eur. J. Biochem. 2002;269:680–687. doi: 10.1046/j.0014-2956.2001.02702.x. PubMed DOI

Piotrowski M. Primary or secondary? Versatile nitrilases in plant metabolism. Phytochemistry. 2008;69:2655–2667. doi: 10.1016/j.phytochem.2008.08.020. PubMed DOI

Howden A.J.M., Preston G.M. Nitrilase enzymes and their role in plant–microbe interactions. Microb. Biotechnol. 2009;2:441–451. doi: 10.1111/j.1751-7915.2009.00111.x. PubMed DOI PMC

Binder B.M. Ethylene signaling in plants. J. Biol. Chem. 2020;295:7710–7725. doi: 10.1074/jbc.REV120.010854. PubMed DOI PMC

Martínková L. Nitrile metabolism in fungi: A review of its key enzymes nitrilases with focus on their biotechnological impact. Fungal Biol. Rev. 2019;33:149–157. doi: 10.1016/j.fbr.2018.11.002. DOI

Basile L.J., Willson R.C., Sewell B.T., Benedik M.J. Genome mining of cyanide degrading nitrilases from filamentous fungi. Appl. Microbiol. Biotechnol. 2008;80:427–435. doi: 10.1007/s00253-008-1559-2. PubMed DOI

Rinágelová A., Kaplan O., Veselá A.B., Chmátal M., Křenková A., Plíhal O., Pasquarelli F., Cantarella M., Martínková L. Cyanide hydratase from Aspergillus niger K10: Overproduction in Escherichia coli, purification, characterization and use in continuous cyanide degradation. Process Biochem. 2014;49:445–450. doi: 10.1016/j.procbio.2013.12.008. DOI

Veselá A.B., Rucká L., Kaplan O., Pelantová H., Nešvera J., Pátek M., Martínková L. Bringing nitrilase sequences from databases to life: The search for novel substrate specificities with a focus on dinitriles. Appl. Microbiol. Biotechnol. 2016;100:2193–2202. doi: 10.1007/s00253-015-7023-1. PubMed DOI

Rucká L., Chmátal M., Kulik N., Petrásková L., Pelantová H., Novotný P., Příhodová R., Pátek M., Martínková L. Genetic and functional diversity of nitrilases in Agaricomycotina. Int. J. Mol. Sci. 2019;20:5990. doi: 10.3390/ijms20235990. PubMed DOI PMC

Basic Local Alignment Search Tool. [(accessed on 30 June 2020)]; Available online: https://blast.ncbi.nlm.nih.gov/Blast.cgi.

Petříčková A., Veselá A.B., Kaplan O., Kubáč D., Uhnáková B., Malandra A., Felsberg J., Rinágelová A., Weyrauch P., Křen V., et al. Purification and characterization of heterologously expressed nitrilases from filamentous fungi. Appl. Microbiol. Biotechnol. 2012;93:1553–1561. Erratum in 2013, 97, 9263–9264. PubMed

Mulelu A.E., Kirykowicz A.M., Woodward J.D. Cryo-EM and directed evolution reveal how Arabidopsis nitrilase specificity is influenced by its quaternary structure. Commun. Biol. 2019;2:260. doi: 10.1038/s42003-019-0505-4. PubMed DOI PMC

Zhang L., Yin B., Wang C., Jiang S., Wang H., Yuan Y.A., Wei D. Structural insights into enzymatic activity and substrate specificity determination by a single amino acid in nitrilase from Synechocystis sp. PCC6803. J. Struct. Biol. 2014;188:93–101. doi: 10.1016/j.jsb.2014.10.003. PubMed DOI

Cuff J.A., Clamp M.E., Siddiqui A.S., Finlay M., Barton G.J. JPred: A consensus secondary structure prediction server. Bioinformatics. 1998;14:892–893. doi: 10.1093/bioinformatics/14.10.892. PubMed DOI

Chen Z., Jiang S., Wang H., Wang L., Wei D. Switching the regioselectivity of two nitrilases toward succinonitrile by mutating the active center pocket key residues through a semi-rational engineering. Chem. Commun. 2019;55:2948–2951. doi: 10.1039/C8CC10110H. PubMed DOI

Howden A.J., Harrison J., Preston G.M. A conserved mechanism for nitrile metabolism in bacteria and plants. Plant J. 2009;57:243–253. doi: 10.1111/j.1365-313X.2008.03682.x. PubMed DOI

Kiziak C., Conradt D., Stolz A., Mattes R., Klein J. Nitrilase from Pseudomonas fluorescens EBC191: Cloning and heterologous expression of the gene and biochemical characterization of the recombinant enzyme. Microbiology. 2005;151:3639–3648. doi: 10.1099/mic.0.28246-0. PubMed DOI

Zhu D., Mukherjee C., Yang Y., Rios B.E., Gallagher D.T., Smith N.N., Biehl E.R., Hua L. A new nitrilase from Bradyrhizobium japonicum USDA 110—Gene cloning, biochemical characterization and substrate specificity. J. Biotechnol. 2008;133:327–333. doi: 10.1016/j.jbiotec.2007.10.001. PubMed DOI

Fernandes B.C.M., Mateo C., Kiziak C., Chmura A., Wacker J., van Rantwijk F., Stolz A., Sheldon R.A. Nitrile hydratase activity of a recombinant nitrilase. Adv. Synth. Catal. 2006;348:2597–2603. doi: 10.1002/adsc.200600269. DOI

Sosedov O., Stolz A. Random mutagenesis of the arylacetonitrilase from Pseudomonas fluorescens EBC191 and identification of variants, which form increased amounts of mandeloamide from mandelonitrile. Appl. Microbiol. Biotechnol. 2014;98:1595–1607. doi: 10.1007/s00253-013-4968-9. PubMed DOI

Xu C., Tang L., Liang Y., Jiao S., Yu H., Luo H. Novel chaperones RrGroEL and RrGroES for activity and stability enhancement of nitrilase in Escherichia coli and Rhodococcus ruber. Molecules. 2020;25:1002. doi: 10.3390/molecules25041002. PubMed DOI PMC

Jamwal S., Dautoo U.K., Ranote S., Dharela R., Chauhan G.S. Enhanced catalytic activity of new acryloyl crosslinked cellulose dialdehyde-nitrilase Schiff base and its reduced form for nitrile hydrolysis. Int. J. Biol. Macromol. 2019;131:117–126. doi: 10.1016/j.ijbiomac.2019.03.034. PubMed DOI

Talley K., Alexov E. On the pH-optimum of activity and stability of proteins. Proteins. 2010;78:2699–2706. doi: 10.1002/prot.22786. PubMed DOI PMC

Jandhyala D., Berman M., Meyers P.R., Sewell B.T., Willson R.C., Benedik M.J. CynD, the cyanide dehydratase from Bacillus pumilus: Gene cloning and structural studies. Appl. Environ. Microbiol. 2003;69:4794–4805. doi: 10.1128/AEM.69.8.4794-4805.2003. PubMed DOI PMC

Effenberger F., Osswald S. Enantioselective hydrolysis of (RS)-2-fluoroarylacetonitriles using nitrilase from Arabidopsis thaliana. Tetrahedron Asymmetry. 2001;12:279–285. doi: 10.1016/S0957-4166(01)00034-9. DOI

Constraint-Based Multiple Alignment Tool. [(accessed on 30 June 2020)]; Available online: https://www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi.

Notredame C., Higgins D.G., Heringa J. T-Coffee: A novel method for fast and accurate multiple sequence alignment. J. Mol. Biol. 2000;302:205–217. doi: 10.1006/jmbi.2000.4042. PubMed DOI

Sali A., Blundell T.L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 1993;234:779–815. doi: 10.1006/jmbi.1993.1626. PubMed DOI

Friesner R.A., Banks J.L., Murphy R.B., Halgren T.A., Klicic J.J., Mainz D.T., Repasky M.P., Knoll E.H., Shelley M., Perry J.K., et al. Glide: A new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J. Med. Chem. 2004;47:1739–1749. doi: 10.1021/jm0306430. PubMed DOI

Schrödinger Release 2018–2. Schrödinger, LLC; New York, NY, USA: 2018.

Krieger E., Darden T., Nabuurs S.B., Finkelstein A., Vriend G. Making optimal use of empirical energy functions: Force-field parameterization in crystal space. Proteins. 2004;57:678–683. doi: 10.1002/prot.20251. PubMed DOI

Krieger E., Dunbrack R.L., Jr., Hooft R.W.W., Krieger B. Assignment of protonation states in proteins and ligands: Combining pKa prediction with hydrogen bonding network optimization. Methods Mol. Biol. 2012;819:405–421. doi: 10.1007/978-1-61779-465-0_25. PubMed DOI

Brunner S., Eppinger E., Fischer S., Gröning J., Stolz A. Conversion of aliphatic nitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191. World J. Microbiol. Biotechnol. 2018;34:91. doi: 10.1007/s11274-018-2477-9. PubMed DOI

Kriechbaumer V., Park W.J., Piotrowski M., Meeley R.B., Gierl A., Glawischnig E. Maize nitrilases have a dual role in auxin homeostasis and β-cyanoalanine hydrolysis. J. Exp. Bot. 2007;58:4225–4233. doi: 10.1093/jxb/erm279. PubMed DOI

MyCurveFit. [(accessed on 7 July 2020)]; Available online: https://mycurvefit.com.

King R.D., Sternberg M.J.E. Identification and application of the concepts important for accurate and reliable protein secondary structure prediction. Protein Sci. 1996;5:2298–2310. doi: 10.1002/pro.5560051116. PubMed DOI PMC

Rost B., Sander C. Prediction of protein secondary structure at better than 70% accuracy. J. Mol. Biol. 1993;232:584–599. doi: 10.1006/jmbi.1993.1413. PubMed DOI

Guermeur Y., Geourjon C., Gallinari P., Deléage G. Improved performance in protein secondary structure prediction by inhomogeneous score combination. Bioinformatics. 1999;15:413–421. doi: 10.1093/bioinformatics/15.5.413. PubMed DOI

Fiser A., Sali A. ModLoop: Automated modeling of loops in protein structures. Bioinformatics. 2003;19:2500–2501. doi: 10.1093/bioinformatics/btg362. PubMed DOI

Canutescu A.A., Dunbrack R.L. Cyclic coordinate descent: A robotics algorithm for protein loop closure. Protein Sci. 2003;12:963–972. doi: 10.1110/ps.0242703. PubMed DOI PMC

Canutescu A.A., Shelenkov A.A., Dunbrack R.L. A graph-theory algorithm for rapid protein side-chain prediction. Protein Sci. 2003;12:2001–2014. doi: 10.1110/ps.03154503. PubMed DOI PMC

Konagurthu A.S., Whisstock J.C., Stuckey P.J., Lesk A.M. MUSTANG: A multiple structural alignment algorithm. Proteins. 2006;64:559–574. doi: 10.1002/prot.20921. PubMed DOI

Willard L., Ranjan A., Zhang H.Y., Monzavi H., Boyko R.F., Sykes B.D., Wishart D.S. VADAR: A web server for quantitative evaluation of protein structure quality. Nucleic Acids Res. 2003;31:3316–3319. doi: 10.1093/nar/gkg565. PubMed DOI PMC

Wiederstein M., Sippl M.J. ProSA-web: Interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res. 2007;35:W407–W410. doi: 10.1093/nar/gkm290. PubMed DOI PMC

Metabolism of Aldoximes and Nitriles in Plant-Associated Bacteria and Its Potential in Plant-Bacteria Interactions