Abstract
Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family.
Highlights
Flavoproteins are a large and diverse family that requires either FMN or FAD as cofactor
The same phylogenetic groups as described before in the analysis of the AtBBE-like enzymes were found in the Brassicaceae [9], if reasonable sub groups of the predefined phylogenetic groups are defined according to sequence distances and active site composition
BBE-like enzymes catalyze a broad range of reactions such as two-electron oxidations as reported for AtBBE-like 15 or four-electron oxidations as shown for Dbv29 [9, 27]
Summary
Flavoproteins are a large and diverse family that requires either FMN or FAD as cofactor. We have shown that two BBE-like enzymes from Arabidopsis thaliana oxidize monolignols to their corresponding aldehydes, i.e. they function as monolignol oxidoreductases [9] This activity suggests that these enzymes are involved in the manipulation of the extracellular monolignol pool and thereby influence plant cell wall metabolism with as yet unknown implications for lignin formation. Monolignol oxidoreductase activity appears to be associated with a defined composition of the active site, as shown in Fig 1 (panel A) Characteristic features of this active site, designated type I, are Tyr117 and Gln438, engaging in a hydrogen bond as well as Tyr479, Tyr193 and Lys436, forming the catalytic base motif. Further inspection of multiple sequence alignments and homology models of BBE-like enzymes revealed the presence of three additional and distinct active site compositions in the remainder of BBE-like enzymes, as shown in Fig 1 (panels B-D, designated type II-IV). By phylogenetic investigations we have elucidated the appearance of BBE-like proteins with a similar active site as AtBBE-like 28 in the plant kingdom
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