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

Abstract

Alginate is a major constituent of mature biofilms produced by Pseudomonas aeruginosa. The penultimate step in the biosynthesis of alginate is the conversion of some β-D-mannuronate residues in the polymeric substrate polymannuronan to α-L-guluronate residues in a reaction catalyzed by C5-mannuronan epimerase. Specificity studies conducted with size-fractionated oligomannuronates revealed that the minimal substrate contained 9 monosaccharide residues. The maximum velocity of the reaction increased from 0.0018 s −1 to 0.0218 s −1 as the substrate size increased from 10 to 20 residues, and no additional increase in k cat was observed for substrates up to 100 residues in length. The K m decreased from 80 μM for substrate containing fewer than 15 residues to 4 μM for substrate containing over 100 residues. In contrast to C5-mannuronan epimerases that have been characterized in other bacterial species, P. aeruginosa C5-mannuronan epimerase does not require Ca 2+ for activity, and the Ca 2+ -alginate complex is not a substrate for the enzyme. Analysis of purified, active enzyme by inductively coupled plasma-emission spectroscopy revealed that no metals were present in the protein. The pH dependence of the kinetic parameters revealed that 3 residues on the enzyme which all have a pK a of about 7.6 must be protonated for catalysis to occur. The composition of the polymeric product of the epimerase reaction was analyzed by 1 H-NMR spectroscopy, which revealed that tracts of adjacent guluronate residues were readily formed. The reaction reached an apparent equilibrium when the guluronate composition of the polymer was 75%.Pseudomonas aeruginosa is an opportunistic human pathogen that has been linked to certain pathological conditions of immunocompromised humans, and especially the respiratory tract infections that accompany cystic fibrosis (1) . Pulmonary infections caused by the mucoid phenotype of P. aeruginosa are almost impossible to eradicate, even with antibiotic treatment (2). The high antibiotic resistance is attributed in part to the formation of a biofilm, a complex extracellular polymeric matrix in which the cells are embedded. One of the primary constituents of the mature P. aeruginosa biofilm is the polysaccharide alginate. This cell-associated virulence factor is a high molecular weight (500-2000 kDa) linear polysaccharide comprised of residues of β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G), which are covalently linked by β-1,4 glycosidic bonds (3). The relative ratio of these building blocks in the polymer and the linear distribution of G residues strikingly alters physical properties of alginate such as viscosity and gel forming ability, and therefore plays a crucial role in the function of the biopolymer (4).Most of the genes required for alginate biosynthesis are located in the algD operon on the P. aeruginosa chromosome (5). The first polymeric product in the pathway is polymannuronan, which is synthesized from GDP-mannuronic acid. The formation of GDP-mannuronic acid from fructose...

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

“…A fixed time coupled assay for AlgG, modified from Jerga et al (31), was used to determine the activity of wild-type AlgG and its mutant variants. In the first step, 4 mg of poly(M) and 500 g of AlgG were mixed in 300 l of buffer C and incubated at 310 K for 40 h. AlgG and its mutant variants were subsequently inactivated by heating the mixture to 373 K for 10 min.…”

“…Therefore, the other residues in the active site region of the D317A mutant have for clarity not been depicted. alginate epimerases use a similar reaction mechanism and because the AlgG epimerization reaction was found to be Ca 2ϩ -independent, then it is likely that AlgG uses an arginine to neutralize the negative charge of the uronic acid (31). Examination of the AlgG structure suggests that the most likely candidate for this role is Arg 345 (Fig.…”

“…and A. vinelandii is AlgG (26 -29). These epimerases share ϳ60% sequence identity and are Ca 2ϩ -independent with a pH optimum for activity of between pH 6 and 7.5 (27,30,31).…”

Identification and Characterization of anAzotobacter vinelandiiType I Secretion System Responsible for Export of the AlgE-Type Mannuronan C-5-Epimerases

“…The AlgG-type MEs are believed not to form G blocks, as alginates isolated from Pseudomonas species are devoid of such structural units. However, recently it was shown that Pseudomonas aeruginosa AlgG can form G blocks in vitro (30). An enzyme homologous to the Pseudomonas AlgG is also encoded in the A. vinelandii genome (48), but its epimerization pattern has not been experimentally determined, due to low in vitro activity.…”

Activated Sugar Precursors: Biosynthetic Pathways and Biological Roles of an Important Class of Intermediate Metabolites in Bacteria

“…GDP-D-mannuronic acid is the activated sugar precursor for alginate polymerization in P. aeruginosa, which is a partially O-acetylated linear polymer of D-mannuronic acid and Lguluronic acid, linked via -1,4 glycosidic bonds (Shankar et al, 1995). In the case of the P. aeruginosa alginate, after polymerization, some D-mannuronic acid residues can be further converted to L-guluronic acid by the extracellular enzyme activity polymannuronic acid C-5-epimerase (Jerga et al, 2006). P. aeruginosa is able to cause severe and life-threatening infections in immunosuppressed patients, such as burn and cancer chemotherapy patients (Wagner and Iglewski, 2008), as well as in patients suffering from cystic fibrosis (CF).…”