Sphingomonas paucimobilis β-glucosidase Bgl1:: a member of a new bacterial subfamily in glycoside hydrolase family 1

The Sphingomonas pauchnobilis beta-glucosidase Bg11 is encoded by the bg11 gene, associated with an 1308 bp open reading frame. The deduced protein has a potential signal peptide of 24 amino acids in the N-terminal region, and experimental evidence is consistent with the processing and export of the Bg11 protein through the inner membrane to the periplasmic space. A His(6)-tagged 44.3 kDa protein was over-produced in the cytosol of Escherichia coli from a recombinant plasmid, which contained the S. paucimobilis bg11 gene lacking the region encoding the putative signal peptide. Mature beta-glucosidase Bg11 is specific for aryl-beta-glucosides and has no apparent activity with oligosaccharides derived from cellulose hydrolysis and other saccharides. A structure-based alignment established structural relations between S. paucimobilis Bg11 and other members of the glycoside hydrolase (GH) family I enzymes. At subsite -1, the conserved residues required for catalysis by GHI enzymes are present in Bg11 with only minor differences. Major differences are found at subsite + 1, the aglycone binding site. This alignment seeded a sequence-based phylogenetic analysis of GH I enzymes, revealing an absence' of horizontal transfer between phyla. Bootstrap analysis supported the definition of subfamilies and revealed that Bg11, the first characterized beta-glucosidase from the genus Sphingonionas, represents a very divergent bacterial subfamily, closer to archaeal subfamilies than to others of bacterial origin.