Recipient Organization
UNIVERSITY OF CALIFORNIA, IRVINE
(N/A)
IRVINE,CA 92697
Performing Department
(N/A)
Non Technical Summary
A. Simple water additives may prevent soft rot damage of plants and crops. B. Simple water additives may help control fruit ripening for purposes of flavor enhancement and transportation. Pectate lyases are proteins that are secreted by plant virulence organisms that cause soft rot diseases. By understanding how pectate lysases cause crop damage, it may be possible to prevent soft-rot by a simple additive to water. Proteins involved in fruit ripening are similar to pectate lyases. By understanding the molecular mechanisms, it may be possible to control fruit ripening better for flavor enhancement and transportation purposes.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Pectate lyases are virulence factors that degrade the alpha(1,4)polygalacturonic acid (PGA) component of plant cell walls, initiating soft rotting diseases in plants. The enzymes are also known to liberate oligosaccharides from higher plant cell walls that function as elicitors of active plant defense reactions. Several studies have indicated that there are sequence and structural plant homologues, for which the role in plant development has not been fully elucidated. The objective of the present proposal is to better understand how pectate lyases and plant homologues function in the plant cell wall. The proposal will seek to understand the requisite atomic features that determine the conditions under which a pectate lyase can also function as a hydrolase. The specific aims requested for the three year grant period are listed below. 1. Characterize the Hydrolase Activity of PelC Mutants Thirty two mutants of Erwinia chrysanthemi pectate lyase C (PelC) have been prepared
and characterized with respect to pectinolytic activity, tissue maceration, and elicitor activity. The PelC mutants will now be characterized with respect to the recently discovered hydrolase activity of PelC. The amino acids that participate in hydrolytic activity will be identified and compared to the set of catalytic amino acids involved in lyase activity. 2. Solve Structure of a PelC Mutant Oligosaccharide Hydrolase Intermediate Based on results of the hydrolytic assays, PelC mutants that are inactive with respect to hydrolase activity will be cocrystallized with oligogalacturonic acid fragments to trap a reaction intermediate. The three dimensional structure of a complex will be solved by crystallographic techniques to visualize the atomic arrangement of the catalytic site. 3. Complete Structure of Catalytic Subunit of Tomato Polygalacturonase The three dimensional structural analysis of the catalytic subunit of tomato polygalacturonase, called PG2, is underway. Crystals have
been grown, xray diffraction data to a resolution of 1.9 Angstroms have been collected, and a molecular replacement solution found. Model construction and refinement will be completed. The active site regions of PG2 and PelC will be compared and analyzed in detail.
Project Methods
1. Biochemical Characterization of Hydrolase Activity of PelC Mutants Thirty two PelC mutants have been prepared and characterized with respect to maceration, lyase, and elicitor activities, but not with respect to the hydrolase activity. The PelC mutants will be tested to determine which, if any, have reduced hydrolase activity. Of the 32 mutants, 15 fall within the pectate lyase active site region and are the primary candidates to test. These include D129N, D131E, D131N, E166D, E166Q, D170N, K172H, K172L, K172R, K190A, R218A, R218E, R218K, R218L, and R223A. K172L and K172H have already been tested, showing increased hydrolase activity as compared to wildtype PelC. The remaining mutants will be freshly purified from the available mutant clones and their hydrolase activity at pH 5.75 will be characterized in the presence and absence of exogenous calcium using the procedures and collaborative assistance, if necessary, of Hotchkiss and colleagues. Not only will the
characterization shed light on the amino acids important for hydrolase activity, but the results should also indicate which mutants are hydrolase-inactive and thus may be useful for trapping a hydrolase intermediate in a crystal structure. 2. Structural Analysis of a PelC Mutant-Oligosaccharide Hydrolase Intermediate The PI will prepare and solve the crystal structure of penta(GalpA) complexed to a PelC mutant that is inactive with respect to hydrolase activity. Based on current knowledge, the most likely PelC mutant to be hydrolase inactive is E166D. Xray diffraction quality crystals of E166D have been grown and once it is determined that E166D is hydrolytically impaired, pentaGalpA will be diffused into the crystals, using the same techniques that led to the successful preparation of the R218K(calcium)pentaGalpA complex crystals. One difference will be the pH of the experiment. To avoid cleavage of the pentaGalpA fragment by the lyase activity of PelC, the pH of the crystal
diffusion experiment will be 5.0. All xray diffraction data will be collected at the ALS synchrotron facility at ALS, where UCI owns part of a beamline. Data processing and computer analysis will be carried out on SGI workstations within the PI's laboratory. 3. Completion of the Structure of the Tomato Ripening Factor, PG2 The structural analysis of tomato PG2 at a resolution of 1.9 Angstrom is well underway. The electron density maps have been phased by the molecular replacement solution using a model partially constructed from AnPGII. The PG2 model construction will be completed and refined using CNS. The PG2 structure will be compared to the four known PG structures from phytopathogenic organisms to determine if the plant homologue differs in some significant way. The PG2 structure will be superimposed upon the R218K(calcium)pentaGalpA structure to identify the similarities and difference of the active site region and to identify the probable locations of the GalpA subsites. The
subsite positions will be verified by diffusing monomer GalpA into the crystals and solving by difference Fourier analysis.