Vanillyl alcohol oxidase from Diplodia corticola: Residues Ala420 and Glu466 allow for efficient catalysis of syringyl derivatives

EGGERICHS, DANIEL; WEINDORF, NILS; MASCOTTI, MARIA LAURA; WELZEL, NATALIE; FRAAIJE, MARCO W.; TISCHLER, DIRK

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Año: 2023

0021-9258

Vanillyl alcohol oxidases belong to the 4-phenol oxidases family and are found predominantlyin lignin-degrading ascomycetes. Systematical investigation of the enzyme family at the sequence level resulted in discovery and characterization of the second recombinantly producedVAO member, DcVAO, from Diplodia corticola. Remarkably high activities for 2,6-substitutedsubstrates like 4-allyl-2,6-dimethoxy-phenol (3.5 ± 0.02 U mg-1) or 4-(hydroxymethyl)-2,6-dimethoxyphenol (6.3 ± 0.5 U mg-1) were observed which could be attributed to a Phe to Alaexchange in the catalytic center. In order to rationalize this rare substrate preference amongVAOs, we resurrected and characterized three ancestral enzymes and performed mutagenesisanalyses. The results indicate that a Cys/Glu exchange was required to retain activity for ɣ-hydroxylations and shifted the acceptance towards benzyl ethers (up to 4.0 ± 0.1 U mg-1). Ourfindings contribute to the understanding of the functionality of VAO enzyme group, and withDcVAO, we add a new enzyme to the repertoire of ether cleaving biocatalysts.