Structural and biochemical studies elucidate the mechanism of rhamnogalacturonan lyase from Aspergillus aculeatus.

Autor: Jensen MH; Biophysical Chemistry Group, Department of Chemistry, University of Copenhagen, 2100 Copenhagen Ø, Denmark., Otten H, Christensen U, Borchert TV, Christensen LL, Larsen S, Leggio LL
Jazyk: angličtina
Zdroj: Journal of molecular biology [J Mol Biol] 2010 Nov 19; Vol. 404 (1), pp. 100-11. Date of Electronic Publication: 2010 Sep 17.
DOI: 10.1016/j.jmb.2010.09.013
Abstrakt: We present here the first experimental evidence for bound substrate in the active site of a rhamnogalacturonan lyase belonging to family 4 of polysaccharide lyases, Aspergillus aculeatus rhamnogalacturonan lyase (RGL4). RGL4 is involved in the degradation of rhamnogalacturonan-I, an important pectic plant cell wall polysaccharide. Based on the previously determined wild-type structure, enzyme variants RGL4_H210A and RGL4_K150A have been produced and characterized both kinetically and structurally, showing that His210 and Lys150 are key active-site residues. Crystals of the RGL4_K150A variant soaked with a rhamnogalacturonan digest gave a clear picture of substrate bound in the -3/+3 subsites. The crystallographic and kinetic studies on RGL4, and structural and sequence comparison to other enzymes in the same and other PL families, enable us to propose a detailed reaction mechanism for the β-elimination on [-,2)-α-l-rhamno-(1,4)-α-d-galacturonic acid-(1,-]. The mechanism differs significantly from the one established for pectate lyases, in which most often calcium ions are engaged in catalysis.
(Copyright © 2010 Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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