The monolignol oxidoreductase AtBBE-like protein 15: Characterization, rational engineering and biocatalytic applications.

Flavoproteins are a diverse protein class employing an isoalloxazine ring for catalysis in form of the flavin mononucleotide (FMN) or the flavin adenine dinucleotide (FAD). Among them is the berberine bridge enzyme-like (BBE-like) protein family 1. Monolignol oxidoreductases from this protein family catalyze the oxidation of monolignols to the corresponding aldehydes 2. Recently we explored the potential of a monolignol oxidoreductase from Arabidopsis thaliana (AtBBE-like protein 15) as bio-catalyst for oxidative reactions. For this study we employed a variant with enhanced reactivity towards oxygen, which was obtained by a single amino acid exchange (L182V)3. The pH and temperature optima of the purified AtBBE-like protein 15 L182V were determined as well as the tolerance toward organic co-solvents; furthermore the substrate scope was characterized. The enzyme has a temperature optimum of 50°C and retains more than 50% activity between pH 5 and pH 10 within 5 min. The enzyme shows increased activity in the presence of various co-solvents (10–50% v/v. Primary benzylic and primary or secondary allylic alcohols were accepted as substrates. The enantioselectivity in the oxidation of secondary alcohols was good to excellent (E>34 to >200). As the enzyme was found to be a versatile and robust biocatalyst we expanded our mutagenesis program to elucidate the catalytic mechanism (presented at the same conference by Julia Messenlehner), to broaden the substrate scope and to alter the enantioselectivity. 1.Daniel, B. et al. The family of berberine bridge enzyme-like enzymes: A treasure-trove of oxidative reactions. Arch. Biochem. Biophys. 632, (2017).2.Daniel, B. et al. Oxidation of Monolignols by Members of the Berberine Bridge Enzyme Family Suggests a Role in Cell Wall Metabolism. J. Biol. Chem. 290, 18770–18781 (2015).3.Pils, S. et al. Characterization of the monolignol oxidoreductase AtBBE-like protein 15 L182V for biocatalytic applications. J. Mol. Catal. B Enzym. (2016). doi:10.1016/j.molcatb.2016.10.018