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 fluorescens subsp. cellulosa polysaccharidases, consists of a Type II cellulose-binding 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, expressed as a discrete entity (CBD(X)) or fused to the catalytic domain (Pf Xyn10A'), bound to amorphous and bacterial microcrystalline cellulose with a K(a) of 2.5 x 10(5) M(-1). CBD(X) exhibited no affinity for soluble forms of cellulose or cello-oligosaccharides, suggesting that the domain interacts with multiple cellulose chains in the insoluble forms of the polysaccharide. Pf Xyn10A' was 2-3 times more active against cellulose-hemicellulose complexes than Pf Xyn10A(CD); however, Pf Xyn10A' and Pf Xyn10A(CD) exhibited the same activity against soluble substrates. CBD(X) did not disrupt the structure of plant-cell-wall material or bacterial microcrystalline cellulose, and did not potentiate Pf Xyn10A(CD) when not covalently linked to the enzyme. There was no substantial difference in the affinity of full-length Pf Xyn10A and 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 Xyn10A(CD) via a serine-rich linker sequence [Bolam, Cireula, McQueen-Mason, Simpson, Williamson, Rixon, Boraston, Hazlewood and Gilbert (1998) Biochem J. 331, 775-781]. These data indicate that CBD(X) is functional in Pf Xyn10A and that no synergy, either in ligand binding or in the potentiation of catalysis, is evident between the Type II and X CBDs of the xylanase.
Gill
J
J
Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, U.K.
Rixon
J E
JE
Bolam
D N
DN
McQueen-Mason
S
S
Simpson
P J
PJ
Williamson
M P
MP
Hazlewood
G P
GP
Gilbert
H J
HJ
D001483:Base Sequence; D001665:Binding Sites; D002482:Cellulose / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D043364:Endo-1,4-beta Xylanases; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014990:Xylans / Q000378:metabolism; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D014990:Xylans; D002482:Cellulose; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases