1b30
From Proteopedia
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XYLANASE FROM PENICILLIUM SIMPLICISSIMUM, COMPLEX WITH 1,2-(4-DEOXY-BETA-L-THREO-HEX-4-ENOPYRANOSYLURONIC ACID)-BETA-1,4-XYLOTRIOSE)
Overview
Following a recent low-temperature crystal structure analysis of the, native xylanase from Penicillium simplicissimum [Schmidt et al. (1998), Protein Sci. 7, 2081-2088], where an array of glycerol molecules, diffused, into the crystal during soaking in a cryoprotectant, was observed within, the active-site cleft, we utilized monomeric xylose as well as a variety, of linear (Xn, n = 2 to 5) and branched xylooligomers at high, concentrations (typically 20% w/v) as cryoprotectant for low-temperature, crystallographic experiments. Binding of the glycosidic moiety (or its, hydrolysis products) to the enzyme's active-site cleft was observed after, as little as 30 s soaking of a native enzyme crystal. The use of a, substrate or substrate analogue as cryoprotectant therefore suggests, itself as a simple and widely applicable alternative to the use of, crystallographic flow-cells for substrate-saturation experiments., Short-chain xylooligomers, i.e., xylobiose (X2) and xylotriose (X3), were, found to bind to the active-site cleft with its reducing end, hydrogen-bonded to the catalytic acid-base catalyst Glu132. Xylotetraose, (X4) and -pentaose (X5) had apparently been cleaved during the soaking, time into a xylotriose plus a monomeric (X4) or dimeric (X5) sugar. While, the trimeric hydrolysis product was always found to bind in the same way, as xylotriose, the monomer or dimer yielded only weak and diffuse electron, density within the xylan-binding cleft, at the opposite side of the active, center. This suggests that the two catalytic residues divide the binding, cleft into a "substrate recognition area" (from the active site toward the, nonreducing end of a bound xylan chain), with strong and specific xylan, binding and a "product release area" with considerably weaker and less, specific binding. The size of the substrate recognition area (3-4 subsites, for sugar rings) explains enzyme kinetic data, according to which short, oligomers (X2 and X3) bind to the enzyme without being hydrolyzed.
About this Structure
1B30 is a Single protein structure of sequence from Penicillium simplicissimum. Active as Endo-1,4-beta-xylanase, with EC number 3.2.1.8 Full crystallographic information is available from OCA.
Reference
Xylan binding subsite mapping in the xylanase from Penicillium simplicissimum using xylooligosaccharides as cryo-protectant., Schmidt A, Gubitz GM, Kratky C, Biochemistry. 1999 Feb 23;38(8):2403-12. PMID:10029534
Page seeded by OCA on Tue Nov 20 11:18:52 2007
