Functional Analyses of Resurrected and Contemporary Enzymes Illuminate an Evolutionary Path for the Emergence of Exolysis in Polysaccharide Lyase Family 2

Abstract

Family 2 polysaccharide lyases (PL2s) preferentially catalyze the β-elimination of homogalacturonan using transition metals as catalytic cofactors. PL2 is divided into two subfamilies that have been generally associated with secretion, Mg(2+) dependence, and endolysis (subfamily 1) and with intracellular localization, Mn(2+) dependence, and exolysis (subfamily 2). When present within a genome, PL2 genes are typically found as tandem copies, which suggests that they provide complementary activities at different stages along a catabolic cascade. This relationship most likely evolved by gene duplication and functional divergence (i.e. neofunctionalization). Although the molecular basis of subfamily 1 endolytic activity is understood, the adaptations within the active site of subfamily 2 enzymes that contribute to exolysis have not been determined. In order to investigate this relationship, we have conducted a comparative enzymatic analysis of enzymes dispersed within the PL2 phylogenetic tree and elucidated the structure of VvPL2 from Vibrio vulnificus YJ016, which represents a transitional member between subfamiles 1 and 2. In addition, we have used ancestral sequence reconstruction to functionally investigate the segregated evolutionary history of PL2 progenitor enzymes and illuminate the molecular evolution of exolysis. This study highlights that ancestral sequence reconstruction in combination with the comparative analysis of contemporary and resurrected enzymes holds promise for elucidating the origins and activities of other carbohydrate active enzyme families and the biological significance of cryptic metabolic pathways, such as pectinolysis within the zoonotic marine pathogen V. vulnificus.

Keywords: enzyme kinetics; enzyme structure; exolysis; magnesium; manganese; polysaccharide lyase; protein evolution.

FIGURE 1.

Function and phylogeny of PL2s. A, β-elimination reaction coordinate resulting in a 4,5-unsaturated product. The dashed semicircles indicate subsites in the positive (toward the reducing end) and negative (toward the non-reducing end) of the scissile bond. B, schematic of exolytic and endolytic modes of activity on HG. Exolytic enzymes digest HG strictly from the terminus of the polysaccharide and release a single defined product. Endolytic enzymes cleave internal glycosidic linkages to generate a mixed product profile. C, representative unrooted tree highlighting the phylogeny of subfamily 1 and 2 members discussed in this study. Boundaries identified by CAZy are indicated with dashed circles. The associated modes of activity are represented with endolytic and exolytic models. The presumed transitional sequence space between these activities is shown with a black triangle. Ye, Y. enterocoliticus; Dd, D. dadantii; Pa, P. atrosepticum; Pae, Paenibacillus sp.

FIGURE 2.

Characterization of HG modification by VvPL2. A, the V. vulnificus HG utilization locus is displayed as a schematic with representative gene sizes shown to scale. Genes that have been classified to assigned CAZy families (CBM, CBM32) and genes predicted in be involved in transport (KdgM and SBP) are labeled. Gene IDs are displayed below. B, product profile of HG digestion by VvPL2 over time. A saturated marker of GalA-GalA₃ is shown on the left, and unsaturated products (degree of polymerization 2–5) are displayed on the right. C, pH profile of VvPL2. The temperature optima of VvPL2 (D), YePL2A (E), and YePL2B (F) are shown. Shown are representative kinetic plots for YePL2A (G), YePL2B (H), and VvPL2 (I) on GalA₃. Product formation represents the detection of ΔGalA by UV absorbance at 232 nm. J, metal exchange assays for YePL2A, YePL2B, and VvPL2 using the dialysis method and quantified using initial velocities with 1 mg ml⁻¹ HG and equivalent enzyme concentrations. The two y axis scales represent product values for YePL2B and VvPL2 (left) and YePL2A (right). Shown are Michaelis-Menten plots for YePL2A (K) and YePL2B (L) in the presence of various catalytic metals using the dialysis method. Error bars, S.D.

FIGURE 3.

Three-dimensional structure of VvPL2. A, schematic model of VvPL2 color-ramped blue (N terminus) to red (C terminus) and with its catalytic metal modeled as a Mn²⁺ shown as a purple sphere. B, superimposition of GalA from the +1 site of the YePL2A complex (Protein Data Bank entry 2V8K) within the active center of VvPL2. The backbone of VvPL2 is shown as a gray schematic with the metal-binding residues displayed as gray sticks, ordered waters as red spheres, Mn²⁺ as a purple sphere, and the stabilizing residue (Arg-304) and Brønstead base (Arg-191) as cyan sticks. The distances between the 2-OH and 3-OH of GalA and Arg-304, C5 and Arg-191, and the Mn²⁺ ion and the uronate group oxygens are labeled and shown as red dashes. C, the metal binding pocket of VvPL2 with N-terminal His tag. The map of the active center residues coordinating the transition metal is presented as maximum likelihood/σ_A weighted 2F_o −F_c densities, contoured at 1.0 σ and carved at 1.5 Å. The coordinated Mn²⁺ and ordered water are displayed as silver and red spheres, respectively. The presence of two tartrate molecules within the N-terminal His tag complex are rendered as yellow ball-and-stick models. D, alignment of the YePL2A (Protein Data Bank entry 2V8J; gray) and VvPL2 (green) metal coordination pocket. Residues are modeled as sticks, Mn²⁺ as purple spheres, and waters as red spheres. Residues are labeled using VvPL2/YePL2A numbering. Bond distances are indicated with yellow dashed lines. Consurf mapping displaying the conserved (magenta) and divergent (cyan) surface features of VvPL2 with subfamily 1 (E) and subfamily 2 (F) members. The highly conserved residues (Lys-152, Arg-191, Arg-304, and metal pocket) and location of the lysine-tryptophan site (K300W) are labeled.

FIGURE 4.

Ancestral sequence reconstruction of the PL2 family. A, phylogeny of the PL2 family with calculated ancestor sequence nodes highlighted (●) and labeled. The node sequences (49, 52, 54, and 74) that were targeted for gene synthesis and biochemical characterization are indicated with a black triangle. Previously characterized enzyme activities are boxed, activities reported in this study are noted with a single asterisk (see Table 1 for references), and family members with solved three-dimensional structures are designated with a double asterisk (3). B, primary structure alignment of YePL2A, YePL2B, VvPL2, Node 52, Node 54, and Node 74. Residues involved in catalysis and metal coordination are indicated with black and white triangles, respectively. The putative lysine to tryptophan switch is highlighted with a black circle.

FIGURE 5.

Product profiling and biochemical characterization of ancestral PL2 sequences. A, high performance anion exchange with pulsed amperometric detection analysis of HG digestion profiles generated by Node 74, Node 52, and Node 54 ancestral enzymes. Pulsed amperometric detection (left axis) of oligogalacturonide elution is displayed as a black trace. The presence of ΔGalA oligosaccharides with a degree of polymerization >2 are indicated with black triangles. B, model for nucleotide progression of lysine-arginine-tryptophan stepwise mutation. C, metal cofactor analysis of Node 74 relative activity with the inset showing the product generation in the presence of Mn²⁺ and Mg²⁺. D, putative subsite structure of PL2 endolytic (top) and exolytic (bottom) active sites displaying the molecular basis of ΔGalA₂ generation by introduction of a tryptophan (black bar). R, reducing end; N, non-reducing end. Brackets indicate terminal residues within the active site that can be extended. Shown are HPLC-pulsed amperometric detection analysis of YePL2B digestions of HG with Trp-286 substitution with lysine and alanine in the absence (E) and presence (F) of EDTA. The large peak after 14 min represents the primary ΔGalA₂ products, and the black arrows indicate the appearance of ΔGalA₃ and ΔGalA₄ products.

FIGURE 6.

Alternative pathways (A and B) for HG utilization by human enteric pathogens that colonize distinct ecosystems. Key differences between the pathways from Y. enterocolitica and V. vulnificus include the following: extracellular, secreted pectin methylesterase (YeCE8) in Y. enterocolitica and pectate lyase (VvPL9) in V. vulnificus; periplasmic, Y. enterocolitica possesses two depolymerases (endoYePL2A and exoYeGH28), and V. vulnificus has only a single enzyme (endoVvPL2); intracellular, intracellular transport appears to be mediated through unrelated systems; cytoplasmic, Y. enterocolitica possesses two exolytic enzymes (exoYePL2B and exoYePL22), whereas V. vulnificus has only one (exoVvPL22). The key conserved features include the KdgM-anionic porin and the HG-binding protein endoCBM32 that is proposed to retain polymerized substrates within the periplasm (3). In Enterobacteriaceae, this cluster exists as a KdgM-endoPL2-CBM32 operon, which is not architecturally conserved in V. vulnificus (Fig. 2A) (18). Enzyme activities that have been biochemically characterized are underlined. #, subfamily for proteins that belong to annotated subfamilies; ×, those families that have not been assigned.