Structural and Biochemical Studies Elucidate the Mechanism of Rhamnogalacturonan Lyase from Aspergillus aculeatus |
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Authors: | Malene H Jensen Harm Otten Torben V Borchert Sine Larsen Leila Lo Leggio |
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Institution: | 1 Biophysical Chemistry Group, Department of Chemistry, University of Copenhagen, 2100 Copenhagen Ø, Denmark2 Novozymes A/S, 2880 Bagsværd, Denmark |
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Abstract: | 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. |
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Keywords: | active site β-elimination polysaccharide lyase family 4 pectin degradation |
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