The family of berberine bridge enzymes in plants

Natural products constitute an immense treasure of chemical compounds with a diverse range of beneficial properties, such as antibiotic, antineoplastic and analgesic activity. Plants are a major source of these compounds with an estimated 25% of currently used medicines derived from plant natural products. However, a great number of plant species are still unexplored and even commonly used model plants (e.g. Arabidopsis thaliana) remain to be investigated with regard to natural products and pathways. Berberine bridge enzyme (BBE)-like enzymes are central catalysts in the biosynthesis of plant alkaloids and terpenes. In recent years, research in our laboratories has led to a detailed understanding of the structure and mechanism of berberine bridge enzyme from the California poppy (Eschscholzia californica). In the proposed study we will extent our research to the 28 genes encoding BBE-like enzymes in the model plant A. thaliana. Recently, we have been able to demonstrate that a member of the BBE-like enzyme family from A. thaliana (termed AtBBE15) catalyses the oxidation of monolignols, such as p-coumaryl alcohol, to their corresponding aldehydes indicating a role of this enzyme in plant cell wall metabolism. Since 14 other members of the BBE-like enzyme family share the same active site composition we suspect that they have similar catalytic properties. Thus one aim of our study will be to demonstrate that these BBE-like enzymes are equally involved in the oxidation of plant monolignols. On the other hand, our analysis has shown that several other groups of BBE-like enzymes are present in A. thaliana that have distinct active sites and thus it is likely that these enzymes play different roles. Therefore the second major aim of our study is to identify the biochemical reactions catalysed by these enzymes and to define their substrate specificity in order to understand their function in planta. Additionally, we will select representatives from each distinct group in the BBE- like family to elucidate the three-dimensional structure of the proteins by means of X-ray crystallography. This effort will be complemented by metabolite analysis of loss- and gain-of- function lines to facilitate identification of potential substrates for the individual BBE-like enzymes. Through this integrative approach of structural biology, biochemistry and plant physiology we expect to define the role of BBE-like enzymes in A. thaliana and we envisage that the gained insights will also promote our understanding of this family of enzymes in plants of medicinal and economical importance.

Berberine bridge enzyme (BBE) is a central enzyme in plant alkaloid biosynthesis catalysing the oxidation of the N-methyl group of (S)-reticuline with concomitant formation of a carbon-carbon bond (the "berberine bridge") to yield (S)-scoulerine. We found that homologs of BBE are widespread among plants, fungi and bacteria. The model plant Arabidopsis thaliana, for example, possesses 28 genes, which apparently encode BBE-like enzymes although the plant does not produce higher alkaloids. In order to reveal the function of BBE-like enzymes from A. thaliana, we investigated the biochemical and structural properties of selected proteins and identified monolignols and their glycosylated derivatives as substrates. Since monolignols are important building blocks for the formation of the plant polymer lignin, we postulate that the BBE-like enzymes of this family play a role in modifying the composition of lignin by oxidizing the monolignols to the corresponding aldehydes ("monolignol oxidoreductases"). This hypothesis was supported by further studies demonstrating the expression of the encoding genes in areas of high and specific lignin production, for example in roots. Since the active site architecture of the monolignol oxidoreductases is conserved in approximately half of the BBE-like enzymes found in the plant kingdom, it appears that this reaction plays an important role in plant life. The diverse biochemical properties of BBE-like enzymes in plants prompted us to look into the original function in basal plants, such as the model moss Physcomitrella patens, which encodes a single BBE-like enzyme (PpBBE). We could show that PpBBE oxidizes the disaccharide cellobiose at the anomeric carbon to yield the corresponding lactone. This is a very interesting finding because many bacterial BBE-like enzymes exhibit related activities and therefore we suggest that the primordial role of BBE-like enzymes in plants is connected to the oxidation of carbohydrates.