Abstract
β-d-Xylosidase from Selenomonas ruminantium is revealed as the best catalyst known (k cat, k cat/K m) for promoting hydrolysis of 1,4-β-d-xylooligosaccharides. 1H nuclear magnetic resonance experiments indicate the family 43 glycoside hydrolase acts through an inversion mechanism on substrates 4-nitrophenyl-β-d-xylopyranoside (4NPX) and 1,4-β-d-xylobiose (X2). Progress curves of 4-nitrophenyl-β-d-xylobioside, xylotetraose and xylohexaose reactions indicate that one residue from the nonreducing end of substrate is cleaved per catalytic cycle without processivity. Values of k cat and k cat/K m decrease for xylooligosaccharides longer than X2, illustrating the importance to catalysis of subsites −1 and +1 and the lack there of subsite +2. Homology models of the enzyme active site with docked substrates show that subsites bey ond−1 are blocked by protein and subsites bey ond +1 are not formed; they suggest that D14 and E186 serve catalysis as general base and general acid, respectively. Individual mutations, D14A and E186A, erode k cat and k cat/K m by <103 and to asimilar extent for substrates 4NPX and 4-nitrophenyl-α-l-arabinofuranoside (4NPA), indicating that the two substrates share the same active site. With 4NPX and 4NPA, pH governs k cat/K m with pK a values of 5.0 and 7.0 assigned to D14 and E186, respectively. k cat (4NPX) has a pK a value of 7.0 and k cat (4NPA) is pH independent above pH 4.0, suggesting that the catalytically inactive, “dianionic” enzyme form (D14-E187-) binds 4NPX but not 4NPA.
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Jordan, D.B., Li, XL., Dunlap, C.A. et al. Structure-function relationships of a catalytically efficient β-D-xylosidase. Appl Biochem Biotechnol 141, 51–76 (2007). https://doi.org/10.1007/s12010-007-9210-8
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DOI: https://doi.org/10.1007/s12010-007-9210-8