Structural and biochemical studies elucidate the mechanism of rhamnogalacturonan lyase from Aspergillus aculeatus.
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.
Jensen
Malene H
MH
Biophysical Chemistry Group, Department of Chemistry, University of Copenhagen, 2100 Copenhagen Ø, Denmark.
Otten
Harm
H
Christensen
Ulla
U
Borchert
Torben V
TV
Christensen
Lars L H
LL
Larsen
Sine
S
Leggio
Leila Lo
LL
D019943:Amino Acid Substitution; D001230:Aspergillus / Q000201:enzymology*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics; D010368:Pectins / Q000737:chemistry / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D005656:Fungal Proteins; D050505:Mutant Proteins; C042491:rhamnogalacturonan I; D010368:Pectins; D011133:Polysaccharide-Lyases