Epimerase Active Domain of Pseudomonas aeruginosa AlgG, a Protein That Contains a Right-Handed β-Helix

Abstract

The polysaccharide alginate forms a protective capsule for Pseudomonas aeruginosa during chronic pulmonary infections. The structure of alginate, a linear polymer of ␤1-4-linked O-acetylated D-mannuronate (M) and L-guluronate (G), is important for its activity as a virulence factor. Alginate structure is mediated by AlgG, a periplasmic C-5 mannuronan epimerase. AlgG also plays a role in protecting alginate from degradation by the periplasmic alginate lyase AlgL. Here, we show that the C-terminal region of AlgG contains a right-handed ␤-helix (RH␤H) fold, characteristic of proteins with the carbohydrate-binding and sugar hydrolase (CASH) domain. When modeled based on pectate lyase C of Erwinia chrysanthemi, the RH␤H of AlgG has a long shallow groove that may accommodate alginate, similar to protein/polysaccharide interactions of other CASH domain proteins. The shallow groove contains a 324-DPHD motif that is conserved among AlgG and the extracellular mannuronan epimerases of Azotobacter vinelandii. Point mutations in this motif disrupt mannuronan epimerase activity but have no effect on alginate secretion. The D324A mutation has a dominant negative phenotype, suggesting that the shallow groove in AlgG contains the catalytic face for epimerization. Other conserved motifs of the epimerases, 361-NNRSYEN and 381-NLVAYN, are predicted to lie on the opposite side of the RH␤H from the catalytic center. Point mutations N362A, N367A, and V383A result in proteins that do not protect alginate from AlgL, suggesting that these mutant proteins are not properly folded or not inserted into the alginate biosynthetic scaffold. These motifs are likely involved in asparagine and hydrophobic stacking, required for structural integrity of RH␤H proteins, rather than for mannuronan catalysis. The results suggest that the AlgG RH␤H protects alginate from degradation by AlgL by channeling the alginate polymer through the proposed alginate biosynthetic scaffold while epimerizing approximately every second D-mannuronate residue to L-guluronate along the epimerase catalytic face.Alginate is a viscous polysaccharide produced by brown seaweed and by certain bacteria, including Pseudomonas and Azotobacter species. Alginate is a high-molecular-weight linear copolymer composed of ␤-D-mannuronic acid (M) and its C 5 epimer ␣-L-guluronic acid (G) linked by ␤1-4 glycosidic bonds. In bacterial but not in algal alginates, the M residues are modified by the addition of O-acetyl groups at the O-2 and/or O-3 position (70).In the opportunistic pathogen Pseudomonas aeruginosa, alginate is an important virulence factor, particularly in patients with the genetic disorder cystic fibrosis, where conversion of strains to the alginate-overproducing (mucoid) phenotype often results in chronic pulmonary P. aeruginosa infections (48). Alginate acts as a virulence factor in these infections by contributing to the matrix material of the mucoid P. aeruginosa biofilms (45) and by protecting the bacteria from opsonic phagocytosis (55). Alginate also neutralizes oxygen r...

Catalytic Mechanism and Mode of Action of the Periplasmic Alginate Epimerase AlgG

“…In vivo studies of P. aeruginosa AlgG revealed that mutagenesis of each of the residues in the conserved DPHD motif (residues 317-320 in P. syringae) abolished epimerase activity (53). This motif is part of the carbohydrate-binding/sugar hydrolysis domain and is proposed to be part of the active site (22,53,55).…”

Structural and Mutational Characterization of the Catalytic A-module of the Mannuronan C-5-epimerase AlgE4 from Azotobacter vinelandii

“…On average, the enzyme epimerizes 10 units ((MG) 10 ) in each reaction before leaving the chain (19). An alignment of all known mannuronan C-5-epimerase sequences from bacteria and algae showed that they share an YG(F/I)DPH(D/E) motif (residues 149 -155 in AlgE4) (21)(22)(23). Asp 152 of this motif, which is also conserved in some pectate lyases, has been shown to be important for activity in AlgE7 (21).…”

Pseudomonas aeruginosa C5-Mannuronan Epimerase: Steady-State Kinetics and Characterization of the Product

“…P. aeruginosa mannuronan epimerase is not closely related to the A. vinelandii enzymes, and although its activity has been reported to be Ca 2+ -dependent, that aspect of the reaction has not been examined in detail. Sequence analysis and homology modeling of the P. aeruginosa epimerase suggests that the epimerase domain of the protein is a right-handed β-helix, which is characteristic of enzymes that utilize polysaccharides as substrates (14). A variety of kinetic studies are presented here, which provide insight into the interactions between the epimerase and alginate.…”