Two crystal structures of pectin lyase A from Aspergillus reveal a pH driven conformational change and striking divergence in the substrate-binding clefts of pectin and pectate lyases.
BACKGROUND
Microbial pectin and pectate lyases are virulence factors that degrade the pectic components of the plant cell wall. The homogalacturan backbone of pectin varies in its degree of methylation from the highly methylated and relatively hydrophobic form known as pectin, to the fully demethylated and highly charged form known as pectate. Methylated and demethylated regions of pectin are cleaved by pectin lyase and calcium-dependent pectate lyases, respectively. Protein engineering of lyases specific for particular patterns of methylation, will yield modified pectins of high value to the food and pharmaceutical industries.


RESULTS
The crystal structures of pectin lyase A from two strains of Aspergillus niger, N400 and 4M-147, have been determined at pH 6.5 (2.4 A resolution) and pH 8.5 (1.93 A resolution), respectively. The structures were determined by a combination of molecular replacement, multiple isomorphous replacement and intercrystal averaging. Pectin lyase A folds into a parallel beta helix and shares many of the structural features of pectate lyases, despite no more than 17% sequence identity after pairwise structure-based alignment. These shared structural features include amino acid stacks and the asparagine ladder. However, the differences in the substrate-binding clefts of these two enzymes are striking. In pectin lyase A, the cleft is dominated by aromatic residues and is enveloped by negative electrostatic potential. In pectate lyases, this cleft is rich in charged residues and contains an elongated ribbon of positive potential when Ca2+ is bound. The major difference between the two pectin lyase A structures from the two strains is in the conformation of the loop formed by residues 182-187. These observed differences are due to the different pH values of crystallization.


CONCLUSIONS
The substrate-binding clefts and catalytic machinery of pectin and pectate lyases have diverged significantly. Specificity is dictated by both the nature of the protein-carbohydrate interaction and long-range electrostatic forces. Three potential catalytic residues have been identified in pectin lyase, two of these are common to pectate lyases. Pectin lyase A does not bind Ca2+ but an arginine residue is found in an equivalent position to the Ca2+ ion in pectate lyase, suggesting a similar role in catalysis. The activity of pectin lyase A is pH -dependent with an optimum activity at pH 5.5. The activity drops above pH 7.0 due to a conformational change at the binding cleft, triggered by the proximity of two buried aspartate residues.
Mayans
O
O
Department of Food Macromolecular Science, Institute of Food Research, Earley Gate, Whiteknights Road, Reading, RG6 6BZ, UK.
Scott
M
M
Connerton
I
I
Gravesen
T
T
Benen
J
J
Visser
J
J
Pickersgill
R
R
Jenkins
J
J
D001234:Aspergillus niger / Q000201:enzymology*; D001665:Binding Sites; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D055672:Static Electricity
D011994:Recombinant Proteins; D011133:Polysaccharide-Lyases; C027349:pectin lyase; C023911:pectate lyase