Oxidation of Monolignols by Members of the Berberine Bridge Enzyme Family Suggests a Role in Plant Cell Wall Metabolism

Bastian Daniel,Tea Pavkov-Keller,Barbara Steiner,Andela Dordic,Alexander Gutmann,Bernd Nidetzky,Christoph W Sensen,Eric Van Der Graaff,Silvia Wallner,Karl Gruber,Peter Macheroux

doi:10.1074/jbc.m115.659631

Abstract

Plant genomes contain a large number of genes encoding for berberine bridge enzyme (BBE)-like enzymes. Despite the widespread occurrence and abundance of this protein family in the plant kingdom, the biochemical function remains largely unexplored. In this study, we have expressed two members of the BBE-like enzyme family from Arabidopsis thaliana in the host organism Komagataella pastoris. The two proteins, termed AtBBE-like 13 and AtBBE-like 15, were purified, and their catalytic properties were determined. In addition, AtBBE-like 15 was crystallized and structurally characterized by x-ray crystallography. Here, we show that the enzymes catalyze the oxidation of aromatic allylic alcohols, such as coumaryl, sinapyl, and coniferyl alcohol, to the corresponding aldehydes and that AtBBE-like 15 adopts the same fold as vanillyl alcohol oxidase as reported previously for berberine bridge enzyme and other FAD-dependent oxidoreductases. Further analysis of the substrate range identified coniferin, the glycosylated storage form of coniferyl alcohol, as a substrate of the enzymes, whereas other glycosylated monolignols were rather poor substrates. A detailed analysis of the motifs present in the active sites of the BBE-like enzymes in A. thaliana suggested that 14 out of 28 members of the family might catalyze similar reactions. Based on these findings, we propose a novel role of BBE-like enzymes in monolignol metabolism that was previously not recognized for this enzyme family.

Highlights

Berberine bridge enzyme-like proteins are a multigene family in plants
Enzymatic Properties and Identification of Substrates of AtBBE-like 13 and AtBBE-like 15—AtBBE-like 13 and AtBBElike 15 were expressed in K. pastoris and purified from the culture medium by nickel-Sepharose affinity chromatography and subsequent gel filtration yielding ϳ10 and 150 mg of protein from 1 liter of fermentation culture, respectively
Because strong protein-ligand interactions are known to increase the thermal stability of proteins, we first screened our library with regard to an increase in the melting temperature of AtBBE-like 15 using the ThermoFluor method [37]

Summary

Background

Berberine bridge enzyme-like proteins are a multigene family in plants. Results: Members of the berberine bridge enzyme-like family were identified as monolignol oxidoreductases. Bridge enzyme (BBE)2-like proteins (pfam 08031) can be set apart due to their unusual bicovalent attachment of the FAD cofactor The namesake of this protein family is BBE from Eschscholzia californica (California poppy) that catalyzes the formation of the so-called “berberine bridge” by oxidation of the N-methyl group of (S)-reticuline yielding (S)-scoulerine [1]. The ␤-O-glycosylated form of coniferyl alcohol (coniferin) is accepted as a substrate Because monolignols and their ␤-glycosylated derivatives (coniferin, syringin, and p-coumaryl-␤-glycoside) are important building blocks and monolignol storage forms, respectively, the catalytic reactions performed by AtBBE-like 13 and AtBBE-like 15 constitute a novel link between the phenylpropanoid pathway and the formation of plant polymerization products, such as lignin and suberin. The characteristic active site found in AtBBE-like 13 and AtBBE-like 15 is conserved in the majority of BBE-like enzymes in A. thaliana (14 of 28) suggesting that they either exhibit different substrate preferences or have distinct spatial (e.g. different plant tissues) or temporal (e.g. in response to pathogens or herbivores) functions in planta

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