Convergent evolution sheds light on the anti-beta -elimination mechanism common to family 1 and 10 polysaccharide lyases

Abstract

Enzyme-catalyzed beta-elimination of sugar uronic acids, exemplified by the degradation of plant cell wall pectins, plays an important role in a wide spectrum of biological processes ranging from the recycling of plant biomass through to pathogen virulence. The three-dimensional crystal structure of the catalytic module of a "family PL-10" polysaccharide lyase, Pel10Acm from Cellvibrio japonicus, solved at a resolution of 1.3 A, reveals a new polysaccharide lyase fold and is the first example of a polygalacturonic acid lyase that does not exhibit the "parallel beta-helix" topology. The "Michaelis" complex of an inactive mutant in association with the substrate trigalacturonate/Ca2+ reveals the catalytic machinery harnessed by this polygalacturonate lyase, which displays a stunning resemblance, presumably through convergent evolution, to the tetragalacturonic acid complex observed for a structurally unrelated polygalacturonate lyase from family PL-1. Common coordination of the -1 and +1 subsite saccharide carboxylate groups by a protein-liganded Ca2+ ion, the positioning of an arginine catalytic base in close proximity to the alpha-carbon hydrogen and numerous other conserved enzyme-substrate interactions, considered in light of mutagenesis data for both families, suggest a generic polysaccharide anti-beta-elimination mechanism.

Fig 1.

(a) Three-dimensional structure of the catalytic domain of Pel10 in divergent (wall-eyed) stereo; (b) Pel1C ligands and the potential base arginine residues in “ball-and-stick” representation, with the Ca²⁺ ions as shaded spheres, and the structures color-ramped from N to C terminus [figure prepared by using MOLSCRIPT/BOBSCRIPT (32, 33)]. (c) Divergent (wall-eyed) stereo surface representation of Pel10Acm with the ligand as licorice, the Ca²⁺ as a sphere, and the guanidinium group of Arg524 shaded blue (prepared by using GRASP 34).

Fig 2.

Observed electron density for the GalA₃/Ca²⁺ complex of Pel10Acm. The map shown is a maximum-likelihood/σ_A weighted 2F_obs − F_calc syntheses contoured at 0.33 electrons per ų. The Pel10A structure is in yellow and the convergent evolution of unrelated pectate lyases revealed by the active-center overlap of R218K mutant of Pel1C shown in dark green. (Arg218 has been reintroduced, in its native location, for reference.) The relative locations of the two putative catalytic base arginines within the protein framework are shown in Fig. 1 a and b for Pel10 and Pel1, respectively.

Fig 3.

Schematic diagram of the interactions of the mutant D389A Pel10Acm with trigalacturonate. The approximate location of Asp389 from the native structure is indicated for reference.

Fig 4.

(a) More O'Ferral diagram for β-elimination of galacto-configured uronic acids. (b) Putative E1cb/asynchronous E2 reaction mechanism for Pel10 and related enzymes in which proton abstraction by arginine is followed by leaving-group elimination. The essential role of Asp389 may involve a role in binding a second Ca²⁺ ion as observed in Pel1C.