Characterization of function and activity of domains A, B and C of xylanase C from Fibrobacter succinogenes S85.
Xylanase C from the ruminant bacterium Fibrobacter succinogenes is comprised of two catalytic domains, A and B, and a third domain, C, of unknown function. The DNA coding for domains A and B of xylanase C were separately cloned and expressed in Escherichia coli as fusion proteins with glutathione-S:-transferase. The fusion proteins were isolated by affinity chromatography on glutathione-Sepharose 4B, cleaved with thrombin and the released xylanase C catalytic domains A and B were purified to apparent homogeneity by anion-exchange chromatography on Mono Q. Electrospray mass spectrometry provided a molecular mass of 27 818 Da (expected, 27 820 Da) for domain B. The pH and temperature optima for activity of domain B on oat spelt xylan were 5.0 and 52 degrees C, respectively. A kinetic analysis of the activity of the catalytic domain A on oat spelt xylan, birch wood xylan and xylooligomers at pH 6.5 and 37 degrees C provided data significantly different to those obtained previously with a protease-derived form of the enzyme [Zhu et al. (1994) J. Bacteriol. 176, 3885-3894]. The isolated domain A was more active on barley-glucan than the protease-derived form and its affinity for birch wood xylan was enhanced resulting in greater overall catalytic efficiency as reflected by k(cat)/K:(M) values. Likewise, significant differences in the Michaelis-Menten parameters K:(M), k(cat) and k(cat)/K:(M) were obtained with domain B compared with values previously reported with this domain attached to domain C. In general, the presence of domain C appeared to decrease the overall efficiency of domain B 7- and 36-fold with birch wood xylan and xylopentaose as substrates, respectively, as reflected by values of k(cat)/K:(M). The removal of domain C also affected the mode of action of domain B such that it more closely resembled that of catalytic domain A. However, no change in either pH and temperature optima or stability were found with domain B compared with the combined domains B and C. The function of domain C remains unknown, but hydrophobic cluster analysis indicated that it may belong to a class of dockerin domains involved in the protein-protein interactions of cellulolytic and xylanolytic complexes.
Marrone
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Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
McAllister
K A
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Clarke
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AJ
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014995:Xylosidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases