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.
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.
Zverlov
V V
VV
Institute of Molecular Genetics, Russian Academy of Science, Kurchatov Sq., 123 182 Moscow, Russia.
Hertel
C
C
Bronnenmeier
K
K
Hroch
A
A
Kellermann
J
J
Schwarz
W H
WH
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology*; D004274:DNA, Recombinant; D004795:Enzyme Stability; D044950:Flavanones; D005419:Flavonoids / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism; D006569:Hesperidin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D013696:Temperature
D004274:DNA, Recombinant; D044950:Flavanones; D005419:Flavonoids; D006027:Glycosides; D006569:Hesperidin; D006026:Glycoside Hydrolases; C022525:alpha-L-rhamnosidase; C005274:naringin