9603805 Yoder The project will study the structural properties of plant virulence factors secreted from bacterial plant pathogens. Two proteins will be studied, polygalacturonase and pectate lyase. The main method to be employed in obtaining structural information will be x-ray diffraction analysis. The proteins chosen for the study include two polygalacturonases and two pectate lyases. The polygalacturonases are Pg1A, from Pseudomonas solanacearum, and Peh1, from Erwinia carotovora. The pectate lyases are PelA from E. chrysanthemi and Pe1153 from E. carotovora. Each protein will be crystallized, and three-dimensional structure determined by x-ray substrate analog will be sought as a means of elucidating amino acids involved in catalysis. Polygalacturonase and pectate lyase both cleave the same saccharide substrate, but by different enzymatic mechanism. Structural comparisons of the two classes of pectinases will be analyzed. The major aims of the proposed four year project are: 1) Purify and crystallize the two polygalacturonases and two pectate lyases. 2) Determine the three-dimensional structure of the pectolytic proteins. It is anticipated that the polygalacturonase structures will be solved using standard multiple isomorphous replacement techniques. The pectate lyase proteins may be amenable to structure determination by molecular replacement techniques. 3) Determine the three-dimensional structure of polygalacturonase bound to inhibitors. Several naturally occurring compounds have been identified as potential inhibitors. 4) Analyze structural differences between polygalacturonases and pectate lyases. Structural components attributing to enzymatic differences will be emphasized. The general question behind the experimental approach for this project is: What are the structural features of two different enzymes--polygalacturonase and pectate lyase--that bind and cleave the same substrate, polygalacturonate, but by different enzymatic mechanisms? The amino acid sequence similarity between these two protein families is very small. Polygalacturonate is the primary component in pectate, an acidic saccharide polymer found in the middle lamella and cell wall of higher plants. The saccharide is important in both cellular adhesion and cell wall integrity. Polygalacturonase cleaves pectate by a hydrolytic mechanism, while pectate lyase cleaves the saccharide by a ( elimination mechanism, requiring calcium for activity. Pectate lyases are structurally unique proteins. Pectate lyase C and E from E. chrysanthemi were the first proteins solved that were composed of a core structure of parallel ( sheets, the folding motif is referred to as a parallel ( helix. The functional implications of this unusual protein fold is unclear. it is unknown if polygalacturonase will adopt the same protein fold as the pectate lyases. The proposed studies will help clarify these questions. The two polygalacturonase proteins will provide a means of comparing the structures of two enzymes with the same enzymatic mechanism, but with different catalytic properties in respect to final endproducts and endproduct inhibition. The structure of PelA will aid in further characterizing the requirements for catalysis and substrate binding with direct comparison to two other known pectate lyase structures from E. chrysanthemi, PelC and PelE. PelA has significantly less enzymatic activity than either PelC or PelE. Pell53 is a pectate lyase that is inefficiently secreted and predominantly localized in the periplasm. A comparison of the structure of Pe1153 to the other Erwinia PL's may be informative in characterizing the targeting signals for secretion.
