Keen 9408999 The three-dimensional structures of two pectate lyase (Pel) proteins have been determined. These proteins are highly stable and are extracellularly secreted by the plant pathogenic bacterium, Erwinia chrysanthemi, to function as key virulence factors. The Pel structures are revolutionary, best described as a 'corkscrew' structure of parallel b strands, not previously observed or even predicted. The parallel b-strands are arranged in a helical structure with ca.22 amino acids per helical turn. The function s of the proteins reside on loops which protrude from the central helix. Since computer algorithms suggest that the parallel b-helix structure may be much more widespread in biology, further characterization of the pectate lyases is lucrative. Our immediate objectives are to characterize the calcium and substrate binding sites of the pectate lyases as well as to determine amino acid residues important in their folding and secretion. Considerable preliminary work has been directed to the development of over- expression systems for pel genes in E.coli and methods for efficiently obtaining oligonucleotide site directed mutations altering one or several specific amino acids. With structural refinement nearing completion for PelC and PelE as well as the ongoing development of fluorescence assays to monitor calcium and substrate binding, the investigators are in position to define mechanisms underlying catalysis, secretion and folding. The results will, in turn, open exciting future avenues. These include critical study of the pectate lyases as pathogen virulence factors; detailed work on their biological secretion; and use of the parallel b-helix as a carrier scaffold for synthetic antigen and catalytic peptide domains that may have utility in clinical medicine. %%% Pectate lyases are important pathogenicity factors released by several pathogens of plants. These enzymatically active proteins attack the integrity of the plant cell wall and per mit pathogen spread in plant tissues and result in damage. As such, these enzymes are a major contributor to the large world-wide loss of food and fiber to disease organisms. Recently, it was shown that a pectate lyases from the pathogenic bacterium, Erwinia chrysanthemi, has a novel structure at the atomic level. This structure, called the parallel beta-helix, is unlike any previously observed structural motif and has important implications on protein structure. The parallel beta-helix structure also has important ramifications for the biological function of pectate lyase in the degradation of plant cells. The present proposal is to study several aspects of the biology of pectate lyase by changing certain amino acids. These changes will be accomplished by techniques involving recombinant DNA technology. This will permit assessing the impact of the changes on the biological properties of the pectate lyase and should eventually allow understanding of the precise interaction of the enzyme with plant tissues. ***
