Functional characterization of the galactan utilization system of Geobacillus stearothermophilus.

UNLABELLED

Type I galactan is a pectic polysaccharide composed of β-1,4 linked units of d-galactose and is part of the main plant cell wall polysaccharides, which are the most abundant sources of renewable carbon in the biosphere. The thermophilic bacterium Geobacillus stearothermophilus T-6 possesses an extensive system for the utilization of plant cell wall polysaccharides, including a 9.4-kb gene cluster, ganREFGBA, which encodes galactan-utilization elements. Based on enzyme activity assays, the ganEFGBA genes, which probably constitute an operon, are induced by short galactosaccharides but not by galactose. GanA is a glycoside hydrolase family 53 β-1,4-galactanase, active on high molecular weight galactan, producing galactotetraose as the main product. Homology modelling of the active site residues suggests that the enzyme can accommodate at least eight galactose molecules (at subsites -4 to +4) in the active site. GanB is a glycoside hydrolase family 42 β-galactosidase capable of hydrolyzing short β-1,4 galactosaccharides into galactose. Applying both GanA and GanB on galactan resulted in the full degradation of the polymer into galactose. The ganEFG genes encode an ATP-binding cassette sugar transport system whose sugar-binding lipoprotein, GanE, was shown to bind galacto-oligosaccharides. The utilization of galactan by G. stearothermophilus involves the extracellular galactanase GanA cleaving galactan into galacto-oligosaccharides that enter the cell via a specific transport system GanEFG. The galacto-oligosaccharides are further degraded by the intracellular β-galactosidase GanB into galactose, which is then metabolized into UDP-glucose via the Leloir pathway by the galKET gene products.

DATABASE

Nucleotide sequence data have been deposited in the GenBank database under the accession number JF327803.