The family of berberine bridge enzyme-like proteins from Arabidopsis thaliana: The family of berberine bridge enzyme-like proteins from Arabidopsis thaliana

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, there is the berberine bridge enzyme-like (BBE-like) protein family (pfam 08031) that was named after the berberine bridge enzyme (EcBBE) from California poppy (Eschscholzia californica). BBE-like proteins form a multigene family in plants and the number of members varies from one in the moss Physcomitrella patens to 57 in the Western Balsam Poplar (Populus trichocarpa). Despite of the frequent occurrence of these proteins their function is largely unknown. Therefore, we chose to investigate the BBE-like proteins occurring in Arabidopsis thalina to broaden our understanding of this protein family. The genome of A. thaliana harbours 28 genes for BBE-like proteins that can be divided into seven phylogenetic groups. Presented are the structural and biochemical characterization of AtBBE-like protein 15 and AtBBE-like protein 28. There are four frequently occurring active site types from AtBBE-like proteins. AtBBE-like 15 is a representative of the most abundant type, which occurs predominantly in dicots. The type found in AtBBE-like 28 is restricted to the plant family Brassicaceae. Additionally, the potential of AtBBE-like protein 15 as biocatalyst was elucidated. This enzyme was identified as a monolignol dehydrogenase, i.e. the enzyme inhibits the reaction between the reduced flavin and oxygen. The enzyme was rationally engineered towards higher oxygen reactivity and the potential of the enzyme as biocatalyst for oxidative reactions was tested. The pH- and temperature optimum (pH 7, 50°C) was determined. The enzyme shows enhanced activity in the presence of various organic solvents and accepts various allylic and benzylic alcohols as substrates. Secondary allylic alcohols were converted with good to excellent enantioselectivity (E>34 to E>200).