110537-88-7Relevant academic research and scientific papers
Glycoside cleavage by a new mechanism in unsaturated glucuronyl hydrolases
Jongkees, Seino A. K.,Withers, Stephen G.
supporting information; experimental part, p. 19334 - 19337 (2012/01/31)
Unsaturated glucuronyl hydrolases (UGLs) from GH family 88 of the CAZy classification system cleave a terminal unsaturated sugar from the oligosaccharide products released by extracellular bacterial polysaccharide lyases. This pathway, which is involved in extracellular bacterial infection, has no equivalent in mammals. A novel mechanism for UGL has previously been proposed in which the enzyme catalyzes hydration of a vinyl ether group in the substrate, with subsequent rearrangements resulting in glycosidic bond cleavage. However, clear evidence for this mechanism has been lacking. In this study, analysis of the products of UGL-catalyzed reactions in water, deuterium oxide, and dilute methanol in water, in conjunction with the demonstration that UGL rapidly cleaves thioglycosides and glycosides of inverted anomeric configuration (substrates that are resistant to hydrolysis by classical glycosidases), provides strong support for this new mechanism. A hydration-initiated process is further supported by the observed UGL-catalyzed hydration of a C-glycoside substrate analogue. Finally, the observation of a small β-secondary kinetic isotope effect suggests a transition state with oxocarbenium ion character, in which the hydrogen at carbon 4 adopts an axial geometry. Taken together, these observations validate the novel vinyl ether hydration mechanism and are inconsistent with either inverting or retaining direct hydrolase mechanisms at carbon 1.
Elucidation of the mechanism of polysaccharide cleavage by chondroitin AC lyase from Flavobacterium heparinum
Rye, Carl S.,Withers, Stephen G.
, p. 9756 - 9767 (2007/10/03)
Chondroitin AC lyase from Flavobacterium heparinum degrades chondroitin sulfate glycosaminoglycans via an elimination mechanism resulting in disaccharides or oligosaccharides with Δ4,5-unsaturated uronic acid residues at their nonreducing end. Mechanistic details concerning the ordering of the bond-breaking and -forming steps of this enzymatic reaction are nonexistent, mainly due to the inhomogeneous nature of the polymeric substrates. The creation of a new class of synthetic substrates for this enzyme has allowed the measurement of defined and reproducible kcat and Km values and has expanded the range of mechanistic studies that can be performed. The primary deuterium kinetic isotope effect upon kcat/Km for the abstraction of the proton α to the carboxylic acid was measured to be 1.67 ± 0.07, showing that deprotonation occurs in a rate-limiting step. Using substrates with leaving groups of differing reactivity, a flat linear free energy relationship was produced, indicating that the C4-O4 bond is not broken in a rate-determining step. Taken together, these results strongly suggest a stepwise mechanism. Consistent with this was the measurement of a secondary deuterium kinetic isotope effect upon kcat/Km of 1.01 ± 0.03 on a 4-{2H}-substrate, indicating that no sp2 character is developed at C4 during the rate-limiting step, thereby ruling out a concerted syn-elimination.
PREPARATION OF PHENYL 4-DEOXY-α- AND β-L-threo-HEX-4-ENOPYRANOSIDURONIC ACIDS AND DETERMINATION OF THE ANOMERIC SPECIFICITY OF THE Δ4,5-GLYCOSIDURONASE INDUCED FROM Flavobacterium heparinum WITH HEPARIN AND CHONDROITIN SULFATE
Ototani, Noboru,Yosizawa, Zensaku
, p. 25 - 36 (2007/10/02)
To investigate the anomeric specificity of the Δ4,5-glycosiduronase induced from Flavobacterium heparinum with heparin and chondroitin sulfate, phenyl 4-deoxy-α- and β-L-threo-hex-4-enopyranosiduronic acids were chemically synthesized and then digested wi
