The aim of this work was to map the sequence space of aldoxime dehydratases (Oxds) as enzymes with great potential for nitrile synthesis. Microbes contain an abundance of putative Oxds but fewer than ten Oxds were characterized in total and only two in fungi. In this work, we prepared and characterized a new Oxd (protein gb|EEU37245.1 named OxdFv) from Fusarium vanettenii 77-13-4. OxdFv is distant from the characterized Oxds with a maximum of 36% identity. Moreover, the canonical Oxd catalytic triad RSH is replaced by R141-E187-E303 in OxdFv. R141A and E187A mutants did not show significant activities, but mutant E303A showed a comparable activity as the wild-type enzyme. According to native mass spectrometry, OxdFv contained almost 1 mol of heme per 1 mol of protein, and was composed of approximately 88% monomer (41.8 kDa) and 12% dimer. A major advantage of this enzyme is its considerable activity under aerobic conditions (25.0 ± 4.3 U/mg for E,Z-phenylacetaldoxime at pH 9.0 and 55 °C). Addition of sodium dithionite (reducing agent) and Fe2+ was required for this activity. OxdFv favored (aryl)aliphatic aldoximes over aromatic aldoximes. Substrate docking in the homology model of OxdFv showed a similar substrate specificity. We conclude that OxdFv is the first characterized Oxd of the REE type.

Institute of Applied Synthetic Chemistry TU Wien Getreidemarkt 9 OC 163 A 1060 Vienna Austria

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

Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 CZ 142 20 Prague Czech Republic; Department of Genetics and Microbiology Faculty of Sciences Charles University Viničná 5 CZ 128 44 Prague Czech Republic

Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 CZ 142 20 Prague Czech Republic; Faculty of Food and Biochemical Technology University of Chemistry and Technology Prague Technická 5 CZ 166 28 Prague Czech Republic

Institute of Molecular Biotechnology Faculty of Technical Chemistry Chemical and Process Engineering Biotechnology Graz University of Technology Petersgasse 14 A 8010 Graz Austria

Institute of Molecular Biotechnology Faculty of Technical Chemistry Chemical and Process Engineering Biotechnology Graz University of Technology Petersgasse 14 A 8010 Graz Austria; Austrian Center of Industrial Biotechnology GmbH Krenngasse 37 A 8010 Graz Austria

Laboratory of Structural Biology and Bioinformatics Institute of Microbiology of the Czech Academy of Sciences Zámek 136 CZ 373 33 Nové Hrady 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; Leibniz Institute of Virology Martinistraße 52 D 20251 Hamburg Germany; European XFEL GmbH Holzkoppel 4 D 22869 Schenefeld Germany

References provided by Crossref.org

Aldoxime dehydratases: production, immobilization, and use in multistep processes

Organic Acid to Nitrile: A Chemoenzymatic Three-Step Route