A recently identified polysaccharide utilisation locus (PUL) from Bacteroides ovatus ATCC8483 is transcriptionally upregulated during growth on galacto- and glucomannans. It encodes two putative glycoside hydrolase family 26 (GH26) β-mannanases, BoMan26A and BoMan26B, and a GH36 α-galactosidase, BoGal36A. The PUL also includes two glycan binding proteins, confirmed by β-mannan affinity electrophoresis. When this PUL was deleted, B. ovatus was no longer able to grow on locust bean galactomannan. BoMan26A primarily formed mannobiose from mannan polysaccharides. BoMan26B had higher activity on galactomannan with a high degree of galactosyl substitution and was shown to be endo-acting generating a more diverse mixture of oligosaccharides including mannobiose. Of the two β-mannanases, only BoMan26B hydrolysed galactoglucomannan. A crystal structure of BoMan26A revealed a similar structure to the exo-mannobiohydrolase CjMan26C from Cellvibrio japonicus, with a conserved glycone region (-1 and -2 subsites) including a conserved loop closing the active site beyond subsite -2. Analysis of cellular location by immuno-labelling and fluorescence microscopy suggests that BoMan26B is surface exposed, associated with the outer membrane, while BoMan26A and BoGal36A are likely periplasmic. In light of the cellular location and the biochemical properties of the two characterised β-mannanases, we propose a scheme of sequential action by the GHs encoded by the β-mannan PUL and involved in the β-mannan utilisation pathway in B. ovatus. The outer membrane associated BoMan26B initially acts on the polysaccharide galactomannan, producing comparably large oligosaccharide fragments. Galactomanno-oligosaccharides are further processed in the periplasm: degalactosylated by BoGal36A and subsequently hydrolysed into mainly mannobiose by the β-mannanase BoMan26A.
|Bidragets titel på inmatningsspråk||Galactomannan catabolism conferred by a polysaccharide utilisation locus of Bacteroides ovatus : enzyme synergy and crystal structure of a β-mannanase |
|Tidskrift||Journal of Biological Chemistry|
|Tidigt onlinedatum||2016 nov 21|
|Status||Published - 2017 jan 6|
- Biokemi och molekylärbiologi
- Annan kemi