Isolation and analysis of a gene encoding alpha-glucuronidase, an enzyme with a novel primary structure involved in the breakdown of xylan.
This is the first report describing the analysis of a gene encoding an alpha-glucuronidase, an enzyme essential for the complete breakdown of substituted xylans. A DNA fragment that carries the gene for alpha-glucuronidase was isolated from chromosomal DNA of the hyperthermophilic bacterium Thermotoga maritima MSB8. The alpha-glucuronidase gene (aguA) was identified and characterized with the aid of nucleotide sequence analysis, deletion experiments and expression studies in Escherichia coli, and the start of the coding region was defined by amino-terminal sequencing of the purified recombinant enzyme. The aguA gene encodes a 674-amino-acid, largely hydrophilic polypeptide with a calculated molecular mass of 78593 Da. The alpha-glucuronidase of T. maritima has a novel primary structure with no significant similarity to any other known amino acid sequence. The recombinant enzyme was purified to homogeneity as judged by SDS-PAGE. Gel filtration analysis at low salt concentrations revealed a high apparent molecular mass (> 630 kDa) for the recombinant enzyme, but the oligomeric structure changed upon variation of the ionic strength or the pH, yielding hexameric and/or dimeric forms which were also enzymatically active. The enzyme hydrolysed 2-O-(4-O-methyl-alpha-D-glucopyranosyluronic acid)-D-xylobiose (MeGlcAX2) to xylobiose and 4-O-methylglucuronic acid. The K(m) for MeGlcAX2 was 0.95 mM. The pH optimum was 6.3. Maximum activity was measured at 85 degrees C, about 25 degrees C or more above the values reported for all other alpha-glucuronidases known to date. When incubated at 55-75 degrees C, the enzyme suffered partial inactivation, but thereafter the residual activity remained nearly constant for several days.
Ruile
P
P
Lehrstuhl für Mikrobiologie, Technische Universität München, Germany.
Winterhalter
C
C
Liebl
W
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial / Q000235:genetics*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D014990:Xylans / Q000378:metabolism*
D014990:Xylans; D006026:Glycoside Hydrolases; C096734:alpha-glucuronidase