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

TY - JOUR

T1 - Structural and biochemical studies elucidate the mechanism of rhamnogalacturonan lyase from Aspergillus aculeatus

AU - Jensen, Malene Hillerup

AU - Otten, Harm

AU - Christensen, Ulla

AU - Borchert, Torben V.

AU - Christensen, Lars L. H.

AU - Larsen, Sine

AU - Lo Leggio, Leila

N1 - Paper id:: doi:10.1016/j.jmb.2010.09.013

PY - 2010/11/19

Y1 - 2010/11/19

N2 - 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.

AB - 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.

KW - Faculty of Science

U2 - 10.1016/j.jmb.2010.09.013

DO - 10.1016/j.jmb.2010.09.013

M3 - Journal article

C2 - 20851126

SN - 0022-2836

VL - 404

SP - 100

EP - 111

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

ER -