{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T22:38:49Z","timestamp":1792795129384,"version":"4.5.0"},"reference-count":34,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,2,3]],"date-time":"2026-02-03T00:00:00Z","timestamp":1770076800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>\n                    Flavoenzymes of the 4-phenol oxidoreductase family are versatile biocatalysts that catalyze the oxidation of a wide variety of phenol derivatives to alcohols, aldehydes, ketones or alkenes. The promiscuous FAD-dependent vanillyl alcohol oxidases from\n                    <jats:italic>Penicillium simplicissimum<\/jats:italic>\n                    (\n                    <jats:italic>Ps<\/jats:italic>\n                    VAO) and\n                    <jats:italic>Diplodia corticola<\/jats:italic>\n                    (\n                    <jats:italic>Dc<\/jats:italic>\n                    VAO) have been described to catalyze the oxidative deamination of\n                    <jats:italic>p<\/jats:italic>\n                    -hydroxybenzylamines, giving rise to valuable flavor compounds, but starting from\n                    <jats:italic>p<\/jats:italic>\n                    -alkyl substituted phenols, the ketones are usually not accessible as these oxidases preferably stop at chiral benzylic alcohols. Here we took a closer look into the fungal VAO family with the aim to identify new members that can perform this deamination reaction and also the overoxidation of benzylic alcohols to ketones at a sufficient rate for application. Phylogenetic and amino acid cluster analysis revealed one clade that differed significantly in the constitution of the active site, while maintaining residues essential for catalysis. From this clade, five candidates were chosen for investigation, which revealed that VAO from\n                    <jats:italic>Paecilomyces variotii<\/jats:italic>\n                    (\n                    <jats:italic>Pv<\/jats:italic>\n                    VAO) showed promising activities with vanillylamine and 4-(1-amino)ethylphenol, especially above pH 9.0, while also offering the ability to perform the overoxidation of\n                    <jats:italic>p<\/jats:italic>\n                    -alkyl substituted phenols toward ketones. Hence, the identified\n                    <jats:italic>Pv<\/jats:italic>\n                    VAO offers two reaction routes toward benzylic ketones.\n                  <\/jats:p>","DOI":"10.3389\/fmicb.2026.1769237","type":"journal-article","created":{"date-parts":[[2026,2,3]],"date-time":"2026-02-03T06:30:33Z","timestamp":1770100233000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["Discovery of a new subfamily expands the catalytic versatility of vanillyl alcohol oxidases"],"prefix":"10.3389","volume":"17","author":[{"given":"Nils","family":"Weindorf","sequence":"first","affiliation":[{"name":"Department of Microbial Biotechnology, Faculty for Biology and Biotechnology, Ruhr University Bochum","place":["Bochum, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tobias","family":"Rapsch","sequence":"additional","affiliation":[{"name":"Department of Microbial Biotechnology, Faculty for Biology and Biotechnology, Ruhr University Bochum","place":["Bochum, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Willem J. H.","family":"van Berkel","sequence":"additional","affiliation":[{"name":"Laboratory of Food Chemistry, Wageningen University & Research","place":["Wageningen, Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dirk","family":"Tischler","sequence":"additional","affiliation":[{"name":"Department of Microbial Biotechnology, Faculty for Biology and Biotechnology, Ruhr University Bochum","place":["Bochum, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,2,3]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"403","DOI":"10.1016\/s0022-2836(05)80360-2","article-title":"Basic local alignment search tool","volume":"215","author":"Altschul","year":"1990","journal-title":"J. Mol. Biol."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"3225","DOI":"10.1002\/cbic.202100457","article-title":"Discovery, biocatalytic exploration and structural analysis of a 4-ethylphenol oxidase from Gulosibacter chungangensis","volume":"22","author":"Alvigini","year":"2021","journal-title":"Chembiochem"},{"key":"ref3","doi-asserted-by":"publisher","first-page":"7865","DOI":"10.1074\/jbc.273.14.7865","article-title":"Molecular cloning, sequencing, and heterologous expression of the vaoA gene from Penicillium simplicissimum CBS 170.90 encoding vanillyl-alcohol oxidase","volume":"273","author":"Benen","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"651","DOI":"10.1111\/j.1432-1033.1992.tb17231.x","article-title":"Purification and characterization of vanillyl-alcohol oxidase from Penicillium simplicissimum. A novel aromatic alcohol oxidase containing covalently bound FAD","volume":"208","author":"De Jong","year":"1992","journal-title":"Eur. J. Biochem."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"171","DOI":"10.1002\/(sici)1097-0290(19980720)59:2<171::aid-bit5>3.0.co;2-e","article-title":"Enantioselective hydroxylation of 4-alkylphenols by vanillyl alcohol oxidase","volume":"59","author":"Drijfhout","year":"1998","journal-title":"Biotechnol. Bioeng."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"110320","DOI":"10.1016\/j.abb.2025.110320","article-title":"Kinetic and structural investigation of the 4-allyl syringol oxidase from Streptomyces cavernae","volume":"765","author":"Eggerichs","year":"2025","journal-title":"Arch. Biochem. Biophys."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"104898","DOI":"10.1016\/j.jbc.2023.104898","article-title":"Vanillyl alcohol oxidase from Diplodia corticola: residues Ala420 and Glu466 allow for efficient catalysis of syringyl derivatives","volume":"299","author":"Eggerichs","year":"","journal-title":"J. Biol. Chem."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s42004-024-01207-1","article-title":"Substrate scope expansion of 4-phenol oxidases by rational enzyme selection and sequence-function relations","volume":"7","author":"Eggerichs","year":"2024","journal-title":"Commun. Chem."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"113277","DOI":"10.1016\/j.mcat.2023.113277","article-title":"Large scale production of vanillin using an eugenol oxidase from Nocardioides sp. YR527","volume":"546","author":"Eggerichs","year":"","journal-title":"Mol. Catal."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"104","DOI":"10.1016\/j.abb.2017.06.022","article-title":"The VAO\/PCMH flavoprotein family","volume":"632","author":"Ewing","year":"","journal-title":"Arch. Biochem. Biophys."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"2546","DOI":"10.1111\/febs.13762","article-title":"A single loop is essential for the octamerization of vanillyl alcohol oxidase","volume":"283","author":"Ewing","year":"2016","journal-title":"FEBS J."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"14668","DOI":"10.1074\/jbc.m117.778449","article-title":"Two tyrosine residues, Tyr-108 and Tyr-503, are responsible for the deprotonation of phenolic substrates in vanillyl-alcohol oxidase","volume":"292","author":"Ewing","year":"","journal-title":"J. Biol. Chem."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"164","DOI":"10.3390\/molecules23010164","article-title":"A Xylenol orange-based screening assay for the substrate specificity of flavin-dependent para-phenol oxidases","volume":"23","author":"Ewing","year":"2018","journal-title":"Molecules"},{"key":"ref14","doi-asserted-by":"publisher","first-page":"435","DOI":"10.1128\/aem.63.2.435-439.1997","article-title":"Enigmatic gratuitous induction of the covalent flavoprotein vanillyl-alcohol oxidase in Penicillium simplicissimum","volume":"63","author":"Fraaije","year":"1997","journal-title":"Appl. Environ. Microbiol."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"18111","DOI":"10.1074\/jbc.272.29.18111","article-title":"Catalytic mechanism of the oxidative demethylation of 4-(methoxymethyl)phenol by vanillyl-alcohol oxidase","volume":"272","author":"Fraaije","year":"1997","journal-title":"J. Biol. Chem."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"43","DOI":"10.1016\/S1381-1177(02)00133-9","article-title":"Covalent flavinylation enhances the oxidative power of vanillyl-alcohol oxidase","volume":"21","author":"Fraaije","year":"2003","journal-title":"J. Mol. Catal. B Enzym."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"712","DOI":"10.1046\/j.1432-1327.1998.2530712.x","article-title":"Kinetic mechanism of vanillyl-alcohol oxidase with short-chain 4-alkylphenols","volume":"253","author":"Fraaije","year":"1998","journal-title":"Eur. J. Biochem."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"38654","DOI":"10.1074\/jbc.m004753200","article-title":"Structural analysis of flavinylation in vanillyl-alcohol oxidase","volume":"275","author":"Fraaije","year":"2000","journal-title":"J. Biol. Chem."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"271","DOI":"10.1111\/j.1432-1033.1995.271_c.x","article-title":"Substrate specificity of flavin-dependent vanillyl-alcohol oxidase from Penicillium Simplicissimum","volume":"234","author":"Fraaije","year":"1995","journal-title":"Eur. J. Biochem."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"285","DOI":"10.1016\/S1389-1723(99)80033-4","article-title":"Purification and characterization of vanillyl-alcohol oxidase from Byssochlamys fulva V107","volume":"87","author":"Furukawa","year":"1999","journal-title":"J. Biosci. Bioeng."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"343","DOI":"10.1038\/nmeth.1318","article-title":"Enzymatic assembly of DNA molecules up to several hundred kilobases","volume":"6","author":"Gibson","year":"2009","journal-title":"Nat. Methods"},{"key":"ref22","doi-asserted-by":"publisher","first-page":"16","DOI":"10.1515\/boca-2017-0002","article-title":"Vanillyl alcohol oxidases produced in Komagataella phaffii contain a highly stable noncovalently bound anionic FAD semiquinone","volume":"3","author":"Gygli","year":"2017","journal-title":"Biocatalysis"},{"key":"ref23","doi-asserted-by":"publisher","first-page":"139","DOI":"10.1016\/S0304-4165(02)00474-9","article-title":"A his-tag based immobilization method for the preparation and reconstitution of apoflavoproteins","volume":"1619","author":"Hefti","year":"2003","journal-title":"Biochim. Biophys. Acta Gen. Subj."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"2311","DOI":"10.1111\/j.1742-4658.2007.05767.x","article-title":"Discovery of a eugenol oxidase from Rhodococcus sp. strain RHA1","volume":"274","author":"Jin","year":"2007","journal-title":"FEBS J."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"907","DOI":"10.1016\/s0969-2126(97)00245-1","article-title":"Crystal structures and inhibitor binding in the octameric flavoenzyme vanillyl-alcohol oxidase: the shape of the active-site cavity controls substrate specificity","volume":"5","author":"Mattevi","year":"1997","journal-title":"Structure"},{"key":"ref26","doi-asserted-by":"publisher","first-page":"1359","DOI":"10.1002\/cbic.201600148","article-title":"Biocatalytic properties and structural analysis of eugenol oxidase from Rhodococcus jostii RHA1: a versatile oxidative biocatalyst","volume":"17","author":"Nguyen","year":"2016","journal-title":"Chembiochem"},{"key":"ref27","doi-asserted-by":"publisher","first-page":"3022","DOI":"10.1093\/molbev\/msab120","article-title":"MEGA11: molecular evolutionary genetics analysis version 11","volume":"38","author":"Tamura","year":"2021","journal-title":"Mol. Biol. Evol."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"4673","DOI":"10.1093\/nar\/22.22.4673","article-title":"CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice","volume":"22","author":"Thompson","year":"1994","journal-title":"Nucleic Acids Res."},{"key":"ref29","doi-asserted-by":"publisher","first-page":"9455","DOI":"10.1073\/pnas.160175897","article-title":"Inversion of stereospecificity of vanillyl-alcohol oxidase","volume":"97","author":"van den Heuvel","year":"","journal-title":"Proc. Natl. Acad. Sci."},{"key":"ref30","doi-asserted-by":"publisher","first-page":"5646","DOI":"10.1128\/jb.180.21.5646-5651.1998","article-title":"Regio- and stereospecific conversion of 4-alkylphenols by the covalent flavoprotein vanillyl-alcohol oxidase","volume":"180","author":"van den Heuvel","year":"1998","journal-title":"J. Bacteriol."},{"key":"ref31","doi-asserted-by":"publisher","first-page":"2954","DOI":"10.1021\/jf010093j","article-title":"Enzymatic synthesis of vanillin","volume":"49","author":"van den Heuvel","year":"2001","journal-title":"J. Agric. Food Chem."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"109","DOI":"10.1016\/S0014-5793(00)01992-X","article-title":"Direction of the reactivity of vanillyl-alcohol oxidase with 4-alkylphenols","volume":"481","author":"van den Heuvel","year":"","journal-title":"FEBS Lett."},{"key":"ref33","first-page":"83","article-title":"Sequence-function relation for the prediction of enzyme properties: a case study on flavin-dependent oxidases","volume-title":"Methods in Enzymology","author":"Weindorf","year":"2025"},{"key":"ref34","doi-asserted-by":"publisher","first-page":"e202405833","DOI":"10.1002\/anie.202405833","article-title":"Chemoenzymatic synthesis of 2-aryl thiazolines from 4-hydroxybenzaldehydes using vanillyl alcohol oxidases","volume":"63","author":"Zhang","year":"2024","journal-title":"Angew. Chem. Int. Ed."}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2026.1769237\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,3]],"date-time":"2026-02-03T06:30:34Z","timestamp":1770100234000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2026.1769237\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,3]]},"references-count":34,"alternative-id":["10.3389\/fmicb.2026.1769237"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2026.1769237","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,3]]},"article-number":"1769237"}}