Functional Implications of the β-Helical Protein Fold: Differences in Chemical and Thermal Stabilities of Erwinia chrysanthemi EC16 Pectate Lyases B, C, and E

Abstract

Colonization of plant tissue by the phytopathogen Erwinia chrysanthemi EC16 is aided by the activities of the pectate lyase isozymes (PLs), which depolymerize the polygalacturonic acid component (PGA) of plant cell walls. The bacterium secretes four pectate lyases (PLa, PLb, PLc, and PLe), two of which, PLc and PLe, have been shown to fold into a similar domain motif, the β-helix. To understand the rationale behind the evolution and retention of these isoforms, the susceptibilities of pectate lyases B, C, and E to chemical and thermal denaturation and the resulting enzymatic inactivation were examined. With guanidine hydrochloride used as a denaturant, all three pectate lyases denatured with transition midpoint guanidine hydrochloride concentrations (Cm) of 1.3, 1.1, and 1.8 M for PLb, PLc, and PLe, respectively. Lyase activity decreased in direct response to loss of secondary structure in all enzymes. Pectate lyases B and C demonstrated increased enzymatic activity at temperatures above 30°C, with maximal activity observed at 40°C for PLb and 35°C for PLc. Transition midpoints (Tm) as measured by circular dichroism were at 46.9°C for PLb and 44.3°C for PLc, indicating detectable conformational changes accompanying thermal inactivation. Decreased enzymatic activity of PLe was observed at all temperatures above 30°C, and the enzyme was found to possess a Tm at 38.9°C. The data demonstrate structural differences among these enzymes that may be the basis for different enzymatic efficiencies under the potential array of environmental conditions experienced by the bacterium. These differences, in turn, may play a part in the retention of these isozymes as virulence factors, allowing the successful colonization of susceptible plant hosts.