Publications

• The metagenome-derived enzyme RhaB opens a new subclass of bacterial B type alpha-L-rhamnosidases.

  Rabausch U., Ilmberger N., Streit W.R.

  A combined sequence- and function-based analysis of a metagenomic library DNA derived from elephant feces led to the identification of a novel bacterial α-l-rhamnosidase belonging to glycoside hydrolase family 78 (GH78). The gene was designated rhaB (4095bp) and encoded for a putative protein of 1 ... >> More

  A combined sequence- and function-based analysis of a metagenomic library DNA derived from elephant feces led to the identification of a novel bacterial α-l-rhamnosidase belonging to glycoside hydrolase family 78 (GH78). The gene was designated rhaB (4095bp) and encoded for a putative protein of 1364 amino acids. The C-terminal part of the enzyme revealed an amino acid (AA) sequence identity of 58% to a predicted bacterial α-l-rhamnosidase from Bacteroides nordii. Interestingly, the N-terminal region of the deduced enzyme RhaB contained a GDSL-like lipase motif and an acetyl-xylan esterase (DAP2) motif. While heterologous expression of the complete rhaB failed, subcloning of the gene identified the most active open reading frame (ORF) to be of 3081bp, which we designated rhaB1. The enzyme RhaB1 was overexpressed in Escherichia coli BL21 (DE3) and was purified to an amount of 75mg/L of culture medium. In accordance to the intestinal origin, RhaB1 showed a preference for mesophilic conditions with an optimum activity at a temperature TOpt of 40°C and a pHOpt of 6.5, respectively. The recombinant protein had a Km value of 0.79mM and a specific activity vmax of 18.4U for pNP-α-l-rhamnose, a calculated Km of 6.36mM and vmax of 2.9×10(-3)U for naringin, and a Km of 6.75mM and specific activity vmax of 8.63×10(-2)U for rutin, respectively. Phylogenetic analysis and amino acid domain architecture comparison revealed that RhaB1 belongs to a new subclass of bacterial B type α-l-rhamnosidases of GH 78. To our knowledge RhaB1 is the first biochemically-characterized enzyme of this subclass. << Less

  J Biotechnol 191:38-45(2014) [PubMed] [EuropePMC]

• The thermostable alpha-L-rhamnosidase RamA of Clostridium stercorarium: biochemical characterization and primary structure of a bacterial alpha-L-rhamnoside hydrolase, a new type of inverting glycoside hydrolase.

  Zverlov V.V., Hertel C., Bronnenmeier K., Hroch A., Kellermann J., Schwarz W.H.

  An alpha-L-rhamnosidase clone was isolated from a genomic library of the thermophilic anaerobic bacterium Clostridium stercorarium and its primary structure was determined. The recombinant gene product, RamA, was expressed in Escherichia coli, purified to homogeneity and characterized. It is a dim ... >> More

  An alpha-L-rhamnosidase clone was isolated from a genomic library of the thermophilic anaerobic bacterium Clostridium stercorarium and its primary structure was determined. The recombinant gene product, RamA, was expressed in Escherichia coli, purified to homogeneity and characterized. It is a dimer of two identical subunits with a monomeric molecular mass of 95 kDa in SDS polyacrylamide gel electrophoresis. At pH 7.5 it is optimally active at 60 degrees C and insensitive to moderate concentrations of Triton X100, ethanol and EDTA. It hydrolysed p-nitrophenyl-alpha-L-rhamnopyranoside, naringin and hesperidin with a specific activity of 82, 1.5 and 0.46 U mg-1 respectively. Hydrolysis occurs by inversion of the anomeric configuration as detected using 1H-NMR, indicating a single displacement mechanism. Naringin was hydrolysed to rhamnose and prunin, which could further be degraded by incubation with a thermostable beta-glucosidase. The secondary structure of RamA consists of 27% alpha-helices and 50% beta-sheets, as detected by circular dichroism. The primary structure of the ramA gene has no similarity to other glycoside hydrolase sequences and possibly is the first member of a new enzyme family. << Less

  Mol Microbiol 35:173-179(2000) [PubMed] [EuropePMC]

• Crystal structure of glycoside hydrolase family 78 alpha-L-Rhamnosidase from Bacillus sp. GL1.

  Cui Z., Maruyama Y., Mikami B., Hashimoto W., Murata K.

  alpha-L-Rhamnosidase (EC 3.2.1.40) catalyzes the hydrolytic release of rhamnose from polysaccharides and glycosides. Bacillus sp. GL1 alpha-L-rhamnosidase (RhaB), a member of glycoside hydrolase (GH) family 78, is responsible for degrading the bacterial biofilm gellan, and also functions as a debi ... >> More

  alpha-L-Rhamnosidase (EC 3.2.1.40) catalyzes the hydrolytic release of rhamnose from polysaccharides and glycosides. Bacillus sp. GL1 alpha-L-rhamnosidase (RhaB), a member of glycoside hydrolase (GH) family 78, is responsible for degrading the bacterial biofilm gellan, and also functions as a debittering agent for citrus fruit in the food and beverage industries through the release of rhamnose from plant glycoside, naringin. The X-ray crystal structure of RhaB was determined by single-wavelength anomalous diffraction using a selenomethionine derivative and refined at 1.9 A resolution with a final R-factor of 18.2%. As is seen in the homodimeric form of the active enzyme, the structure of RhaB in crystal packing is a homodimer containing 1908 amino acids (residues 3-956), 43 glycerol molecules, four calcium ions, and 1755 water molecules. The overall structure consists of five domains, four of which are beta-sandwich structures designated as domains N, D1, D2, and C, and an (alpha/alpha)(6)-barrel structure designated as domain A. Structural comparison by DALI showed that RhaB shares its highest level of structural similarity with chitobiose phosphorylase (Z score of 25.3). The structure of RhaB in complex with the reaction product rhamnose (inhibitor constant, K(i)=1.8 mM) was also determined and refined at 2.1 A with a final R-factor of 19.5%. Rhamnose is bound to the deep cleft of the (alpha/alpha)(6)-barrel domain, as is seen in the clan-L GHs. Several negatively charged residues, such as Asp567, Glu572, Asp579, and Glu841, conserved in GH family 78 enzymes, interact with rhamnose, and RhaB mutants of these residues have drastically reduced enzyme activity, indicating that the residues are crucial for enzyme catalysis and/or substrate binding. To our knowledge, this is the first report on the determination of the crystal structure of alpha-L-rhamnosidase and identification of its clan-L (alpha/alpha)(6)-barrel as a catalytic domain. << Less

  J Mol Biol 374:384-398(2007) [PubMed] [EuropePMC]

• -L-rhamnosidases of the liver of Turbo cornutus and Aspergillus niger.

  Kurosawa Y., Ikeda K., Egami F.

  J Biochem 73:31-37(1973) [PubMed] [EuropePMC]

• Purification and characterization of an alpha-L-rhamnosidase from Pichia angusta X349.

  Yanai T., Sato M.

  An intracellular alpha-L-rhamnosidase from Pichia angusta X349 was purified to homogeneity through four chromatographic steps. The alpha-L-rhamnosidase appeared to be a monomeric protein with a molecular mass of 90 kDa. The enzyme had an isoelectric point at 4.9, and was optimally active at pH 6.0 ... >> More

  An intracellular alpha-L-rhamnosidase from Pichia angusta X349 was purified to homogeneity through four chromatographic steps. The alpha-L-rhamnosidase appeared to be a monomeric protein with a molecular mass of 90 kDa. The enzyme had an isoelectric point at 4.9, and was optimally active at pH 6.0 and at around 40 degrees C. The Ki for L-rhamnose inhibition was 25 mM. The enzyme was inhibited by Cu2+, Hg2+, and p-chloromercuribenzoate. The alpha-L-rhamnosidase was highly specific for alpha-L-rhamnopyranoside and liberated rhamnose from naringin, rutin, hesperidin, and 3-quercitrin. The alpha-L-rhamnosidase was active at the ethanol concentrations of wine. It efficiently released monoterpenols, such as linalool and geraniol, from an aroma precursor extracted from Muscat grape juice. << Less

  Biosci Biotechnol Biochem 64:2179-2185(2000) [PubMed] [EuropePMC]