The type II and X cellulose-binding domains of Pseudomonas xylanase A potentiate catalytic activity against complex substrates by a common mechanism

Xylanase A (Pf Xyn10A), in common with several other Pseudomonas fluorescen s subsp. cellulosa polysaccharidases, consists of a Type II cellulose-bindi ng domain (CBD), a catalytic domain (Pf Xyn10A(CD)) and an internal domain that exhibits homology to Type X CBDs. The Type X CBD of Pf Xyn10A, express ed as a discrete entity (CBDx) or fused to the catalytic domain (Pf Xyn10A' ), bound to amorphous and bacterial microcrystalline cellulose with a K-a o f 2.5 x 10(5) M-1. CBD, exhibited no affinity for soluble forms of cellulos e or cello-oligosaccharides, suggesting that the domain interacts with mult iple cellulose chains in the insoluble forms of the polysaccharide. Pf Xyn1 0A' was 2-3 times more active against cellulose-hemicellulose complexes tha n Pf Xyn10A(CD); however, Pf Xyn10A' and Pf Xyn10A(CD), exhibited the same activity against soluble substrates. CBD, did not disrupt the structure of plant-cell-wall material or bacterial microcrystalline cellulose, and did n ot potentiate Pf Xyn10A(CD) when not covalently linked to the enzyme. There was no substantial difference in the affinity of full-length Pf Xyn 10A an d the enzyme's Type II CBD for cellulose. The activity of Pf Xyn10A against cellulose-hemicellulose complexes was similar to that of Pf Xyn10A', and a derivative of Pf Xyn10A in which the Type II CBD is linked to the Pf Xyn10 A(CD) via a serine-rich linker sequence [Bolam, Cireula, McQueen-Mason, Sim pson, Williamson, Rixon, Boraston, Hazlewood and Gilbert (1998) Biochem J. 331, 775-781]. These data indicate that CBDx is functional in Pf Xyn10A and that no synergy, either in ligand binding or in the potentiation of cataly sis, is evident between the Type II and X CBDs of the xylanase.