Michael L. Sinnott

Definition of the substrate specificity of the 'sensing' xylanase of Streptomyces cyaneus using xylooligosaccharide and cellooligosaccharide glycosides of 3,4-dinitrophenol

The title compounds, (Xylp beta (1-->4))nXylp beta-3,4-DNP (n = 0-4) have been made by selective anomeric deprotection of peracetylated xylose oligosaccharides with hydrazine, followed by formation of the trichloroacetimidate, uncatalysed reaction with 3,4-dinitrophenol, and Zemplén deacetylation. The values of k(cat)/K(m) for 3,4-dinitrophenol release from these substrates by xylanase III of Streptomyces cyaneus, expressed in Escherichia coli, increase with increasing n up to n = 2 and then slightly decrease. Since it is known from previous work that in its normal host, the enzyme is produced constitutively at low levels and excreted, these results suggest that the biological function of the enzyme may be to produce small molecule inducers, predominantly xylotriose, from the non-reducing end of the xylan. Activity on cellooligosaccharide glycosides (Glcp beta (1-->4))nGlcp beta-3,4-DNP (n = 0-3) was detected, at a rate about two-and-a-half orders of magnitude less than that observed on the corresponding xylooligosaccharides, indicating that the enzyme is a true xylanase.

Hydrolysis of glycosylpyridinium ions by anomeric-configuration-inverting glycosidases

The hydrolyses of five beta-D-xylopyranosylpyridinium ions by the beta-D-xylosidase of Bacillus pumilus proceed with kcat values 10(8)-10(9)-fold larger than the rates of spontaneous hydrolysis of the same compounds. Log(kcat) values correlate well with aglycon pK(a) [B1g(V) = -0.52, r = 0.99], whereas the correlation of log(kcat/Km) is poor [r = 0.77; beta 1g(V/K) = approximately -0.6]. The (1-->3)-beta-D-glucanase of Sporotrichum dimorphosporum hydrolyses 4-bromo-2-(beta-D-glucopyranosyl)isoquinolinium ion with a rate enhancement of 10(8). The amyloglucosidase II of Aspergillus niger hydrolyses three alpha-D-glucopyranosylpyridinium ions with rate enhancements of 10(5)-10(8). The efficient hydrolysis of glycosylpyridinium ions by these three inverting glycosidases, the catalytic mechanism of which is unlikely to involve a nucleophile from the enzyme, makes it improbable that the hydrolysis of glycosylpyridinium ions by retaining glycosidases, discovered some years ago, is initiated by addition of a catalytic nucleophilic carboxylate group of the enzyme to the pyridinium ring.