α-L-rhamnosidases: Old and new insights

Abstract

L-Rhamnose is a component of plant cell wall pectic polysaccharides (Mutter et al., 1994; Ridley et al., 2001), glycoproteins (Haruko and Haruko, 1999) and secondary metabolites such as anthocyanins (Renault et al., 1997), flavonoids (Bar-Peled et al., 1991) and triterpenoids (Friedman and McDonald, 1997). It has also been found in bacterial heteropolysaccharides (Hashimoto and Murata, 1998), rhamnolipids (Ochsner et al., 1994) and in the repeating units of the O-antigen structure of the lipopolysaccharide component of bacterial outer membranes (Chua et al., 1999). Some rhamnosides are important bioactive compounds, e.g. cytotoxic saponins (Bader et al., 1998; Yu et al., 2002), antifungal plant glycoalkaloids (Oda et al., 2002) and bacterial virulence factors (Deng et al., 2000). In plants L-rhamnose-containing terpenyl glycosides are important aroma precursors (Gnata et al., 1985) and may also play a protective role against the toxicity of free aglycons; L-rhamnose-containing flavonoid glycosides have antioxidant and antiinflammatory activities (Benavente-García et al., 1997). α-L-rhamnosidases (EC 3.2.1.40) and β-L-rhamnosidases (EC 3.2.1.43) catalyse the hydrolysis of terminal, non-reducing L-rhamnose residues in α- and β-L-rhamnosides respectively. In contrast, endorhamnosidases (EC 3.2.1.-) act by cleaving specific linkages between internal rhamnose residues in rhamnosides. α-L-rhamnosidases have been found in many micro-organisms and in some plant and animal tissues (see below), whereas β-L-rhamnosidase has only been described in Klebsiella aerogenes (Barker et al., 1965). Endorhamnosidases seem to be restricted to bacteriophages (Steinbacher et al., 1994; Chua et al., 1999). This chapter will mainly focus on the α-L-rhamnosidases (αRHAs) as a group of hydrolytic enzymes having crucial biological functions and important potential biotechnological applications. In 1991 a classification of glycoside hydrolases based on amino acid sequence similarities was introduced (Henrissat, 1991). This classification, which is regularly updated, now comprises more than 100 sequence-based families (URL://http://afmb.cnrs-mrs.fr/CAZY/). The αRHAs, with the exception of that of Sphingomonas paucimobilis that has recently been assigned to family 106, belong to family 78 (Coutinho and Henrissat, 1999). Currently, 61 different putative αRHAs from bacteria, yeasts and moulds are known. Genome-sequencing projects, particularly those focussing on bacterial, fungal and plant genomes, are beginning to generate large numbers of potential αRHA sequences, and a very recent search of the NCBI database (http://www.ncbi.nlm.nih.gov) yielded more than 100 entries for putative αRHA encoding genes. Endorhamnosidases, with the exception of that of bacteriophage Sf6 which has not been classified, are placed in family 90. αRHAs are inverting glycoside hydrolases. Enzymatic hydrolysis of the glycosidic bond takes place via general acid catalysis that requires two critical residues: a proton donor and a nucleophile/base (Davies and Henrissat, 1995). These residues are as yet unidentified in αRHAs. In general, inverting glycosyl hydrolases (GH) typically employ two side chain carboxylates (supplied by Asp or Glu) in the active site to effect catalysis. αRHAs have recently been the focus of several research initiatives because of their key roles in fundamental biological processes (e.g. detoxification mechanisms, symbiosis) and utility in biotechnological applications (e.g. elucidation of the structures of biologically important glycosides, biomass conversion, beverage quality enhancement and the manufacture of hydrolysis products from natural glycosides). © 2007 Springer.