X-ray analysis of crystals of polygalacturonase A fromPseudomonas solanacearum

Abstract

Crystals of the pectolytic protein, polygalacturonase A, have been obtained from polyethylene glycol 8000 using vapordiffusion methods. The 52.4 kDa protein is secreted by the plant pathogenic bacteria Pseudomonas solanacearum, and is important in the virulence of this plant pathogen. The protein crystallizes in space group P21 and has unit-cell parameters of a = 101.9, b = 124.6, c = 48.1 A, and/3 = 105o50 '. The crystal has two molecules in the asy.mmetric unit, and diffracts maximally to a resolution of 2.1 A.Polygalacturonases (PG's) are one of a host of enzymes secreted by microbial plant pathogens capable of attacking plant cell components. Pectate-degrading enzymes have been shown to play an important role in pathogenesis by numerous bacterial plant pathogens (Collmer & Keen, 1986). Pseudomonas solanacearum is a soil-borne plant pathogen that causes wilting diseases in many crop plants worldwide (Hayward, 1991). Schell, Roberts & Denny (1988) and Denny, Carney & Schell (1990) were able to demonstrate that a major secreted PG encoded by the pglA gene plays a significant role in disease, but is not absolutely necessary for pathogenesis. P. solanacearum produces at least two other PG's besides PglA, both are apparently exopolygalacturonases (Schell, Denny & Huang, 1994). To facilitate purification and increase yields of PglA for crystallization experiments, the protein was purified from culture supernatants of a strain engineered to overproduce PglA and to have reduced production of other extracellular enzymes. Based on its deduced amino-acid sequence (Huang & Schell, 1990), mature PglA has 508 amino acids, a pl of 8.8, and a molecular mass of 52.4 kDa. The sequence of PglA between residues 309 and 371 has 55% sequence similarity with PG's from tomato and E. carotovora (Schell et al., 1994). The two proteins may not be closely related though, due to differences in the restriction maps of the genes for the PG's from P.solanacearum and E. carotovora, and to differences in the molecular weights of the proteins (Hinton, Gill, Lalo, Plastow & Salmond, 1990).There are two major families of pectolytic enzymes, pectate lyases and polygalacturonases. Although the substrates cleaved by both families of enzymes are similar, their enzymatic mechanisms differ. While pectate lyases cleave by a /3 elimination, PG's cleave by hydrolysis. One of the first reports of crystallization of a pectolytic enzyme was by Uchino, Kurono & Shinji (1966) who reported obtaining microcrystals of a fungal PG from ammonium sulfate during purification. Recently a second report of the crystallization of a fungal polygalacturonase from Aspergillus niger has appeared, and includes a preliminary X-ray crystallographic characterization * Author to whom all correspondence should be addressed.© 1995 International Union of Crystallography Printed in Great Britain -all rights reserved of the protein (Schrtiter, Arkema, Kester, Visser & Dijkstra, 1994). Currently there are three high-resolution structures of pectolytic enzymes reported, all of...