2 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 homogal acturan backbone of pectin varies in its degree of methylation from th e highly methylated and relatively hydrophobic form known as pectin, t o the fully demethylated and highly charged form known as pectate. Met hylated and demethylated regions of pectin are cleaved by pectin lyase and calcium-dependent pectate lyases, respectively. Protein engineeri ng of lyases specific far particular patterns of methylation, will yie ld modified pectins of high value to the food and pharmaceutical indus tries. Results: The crystal structures of pectin lyase A from two stra ins of Aspergillus niger, N400 and 4M-147, have been determined at pH 6.5 (2.4 Angstrom resolution) and pH 8.5 (1.93 Angstrom resolution), r espectively. The structures were determined by a combination of molecu lar replacement, multiple isomorphous replacement and intercrystal ave raging. Pectin lyase A folds into a parallel beta helix and shares man y of the structural features of pectate lyases, despite no more than 1 7% sequence identity after pairwise structure-based alignment. These s hared structural features include amino acid stacks and the asparagine ladder. However, the differences in the substrate-binding clefts of t hese two enzymes are striking. In pectin lyase A, the cleft is dominat ed by aromatic residues and is enveloped by negative electrostatic pot ential. 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 t he two strains is in the conformation of the loop formed by residues 1 82-187. These observed differences are due to the different pH values of crystallization. Conclusions: The substrate-binding clefts and cata lytic machinery of pectin and pectate lyases have diverged significant ly. Specificity is dictated by both the nature of the protein-carbohyd rate 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 i n 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 resi dues.