Epimerase active domain of Pseudomonas aeruginosa AlgG, a protein that contains a right-handed beta-helix

The polysaccharide alginate forms a protective capsule for Pseudomonas aeruginosa during chronic pulmonary infections. The structure of alginate, a linear polymer of beta1-4-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 beta-helix (RHbetaH) fold, characteristic of proteins with the carbohydrate-binding and sugar hydrolase (CASH) domain. When modeled based on pectate lyase C of Erwinia chrysanthemi, the RHbetaH 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 RHbetaH 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 RHbetaH proteins, rather than for mannuronan catalysis. The results suggest that the AlgG RHbetaH 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.     

The dual roles of AlgG in C-5-epimerization and secretion of alginate polymers in Pseudomonas aeruginosa

Pseudomonas aeruginosa strains causing chronic pulmonary infections in cystic fibrosis patients produce high levels of alginate, an exopolysaccharide that confers a mucoid phenotype. Alginate is a linear polymer of d-mannuronate (M) and variable amounts of its C-5-epimer, l-guluronate (G). AlgG is a periplasmic C-5-epimerase that converts poly d-mannuronate to the mixed M+G sequence of alginate. To understand better the role and mechanism of AlgG activity, a mutant was constructed in the mucoid strain FRD1 with a defined non-polar deletion of algG. Instead of producing poly mannuronate, the algG deletion mutant secreted dialysable uronic acids, as does a mutant lacking the periplasmic protein AlgK. High levels of unsaturated ends and the nuclear magnetic resonance spectroscopy pattern revealed that the small, secreted uronic acids were the products of extensive polymer digestion by AlgL, a periplasmic alginate lyase co-expressed with AlgG and AlgK. Thus, AlgG is bifunctional with (i) epimerase activity and (ii) a role in protecting alginate from degradation by AlgL during transport through the periplasm. AlgK appears to share the second role. AlgG and AlgK may be part of a periplasmic protein complex, or scaffold, that guides alginate polymers to the outer membrane secretin (AlgE). To characterize the epimerase activity of AlgG further, the algG4 allele of poly mannuronate-producing FRD462 was shown to encode a protein lacking only the epimerase function. The sequence of algG4 has a Ser-272 to Asn substitution in a serine-threonine-rich and conserved region of AlgG, which revealed a critical residue for C-5-epimerase activity.     

Pseudomonas aeruginosa AlgG is a polymer level alginate C5-mannuronan epimerase.

Alginate is a viscous extracellular polymer produced by mucoid strains of Pseudomonas aeruginosa that cause chronic pulmonary infections in patients with cystic fibrosis. Alginate is polymerized from GDP-mannuronate to a linear polymer of beta-1-4-linked residues of D-mannuronate and its C5-epimer, L-guluronate. We previously identified a gene called algG in the alginate biosynthetic operon that is required for incorporation of L-guluronate residues into alginate. In this study, we tested the hypothesis that the product of algG is a C5-epimerase that directly converts D-mannuronate to L-guluronate. The DNA sequence of algG was determined, and an open reading frame encoding a protein (AlgG) of approximately 60 kDa was identified. The inferred amino terminus of AlgG protein contained a putative signal sequence of 35 amino acids. Expression of algG in Escherichia coli demonstrated both 60-kDa pre-AlgG and 55-kDa mature AlgG proteins, the latter of which was localized to the periplasm. An N-terminal analysis of AlgG showed that the signal sequence was removed in the mature form. Pulse-chase experiments in both E. coli and P. aeruginosa provided evidence for conversion of the 60- to the 55-kDa size in vivo. Expression of algG from a plasmid inan algG (i.e., polymannuronate-producing) mutant of P. aeruginosa restored production of an alginate containing L-guluronate residues. The observation that AlgG is apparently processed and exported from the cytoplasm suggested that it may act as a polymer-level mannuronan C5-epimerase. An in vitro assay for mannuronan C5 epimerization was developed wherein extracts of E. coli expressing high levels of AlgG were incubated with polymannuronate. Epimerization of D-mannuronate to L-guluronate residues in the polymer was detected enzymatically, using a L-guluronate-specific alginate lyase of Klebsiella aerogenes. Epimerization was also detected in the in vitro reaction between recombinant AlgG and poly-D-mannuronate, using high-performance anion-exchange chromatography. The epimerization reaction was detected only when acetyl groups were removed from the poly-D-mannuronate substrate, suggesting that AlgG epimerization activity in vivo may be sensitive to acetylation of the D-mannuronan residues. These results demonstrate that AlgG has polymer-level mannuronan C5-epimerase activity.     

Characterization of Three New Azotobacter vinelandii Alginate Lyases,One of Which Is Involved in Cyst Germination

Alginates are polysaccharides composed of 1-4-linked β-d-mannuronic acid and α-l-guluronic acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a β-elimination reaction. Two such lyases have previously been identified in the soil bacterium Azotobacter vinelandii, as follows: the periplasmic AlgL and the secreted bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7. In this work, we describe the properties of three new lyases from this bacterium, AlyA1, AlyA2, and AlyA3, all of which belong to the PL7 family of polysaccharide lyases. One of the enzymes, AlyA3, also contains a C-terminal module similar to those of proteins secreted by a type I secretion system, and its activity is stimulated by Ca²⁺. All three enzymes preferably cleave the bond between guluronic acid and mannuronic acid, resulting in a guluronic acid residue at the new reducing end, but AlyA3 also degrades the other three possible bonds in alginate. Strains containing interrupted versions of alyA1, alyA3, and algE7 were constructed, and their phenotypes were analyzed. Genetically pure alyA2 mutants were not obtained, suggesting that this gene product may be important for the bacterium during vegetative growth. After centrifugation, cultures from the algE7 mutants form a large pellet containing alginate, indicating that AlgE7 is involved in the release of alginate from the cells. Upon encountering adverse growth conditions, A. vinelandii will form a resting stage called cyst. Alginate is a necessary part of the protective cyst coat, and we show here that strains lacking alyA3 germinate poorly compared to wild-type cells.Azotobacter vinelandii is a nitrogen-fixing bacterium found in soil. A. vinelandii and several species belonging to the related genus Pseudomonas have been found to produce the polymer alginate. This linear, extracellular polysaccharide is composed of 1-4-linked β-d-mannuronic acid (M) and its C-5 epimer α-l-guluronic acid (G) (35), and the relative amount and distribution of these two residues vary according to the species and growth conditions. Some of the M residues in bacterial alginates may be O acetylated at C-2, C-3, or both C-2 and C-3 (34).Alginate is first synthesized as mannuronan, and the G residues are introduced by mannuronan C-5 epimerases. All genome-sequenced alginate-producing bacteria have been found to encode a periplasmic epimerase, AlgG, that epimerizes some of the M residues in the polymer into G residues (40). AlgG seems to be unable to epimerize an M residue next to a preexisting G residue in vivo. A. vinelandii also encodes a family of secreted mannuronan C-5 epimerases (AlgE1-7) (40), some of which are able to form stretches of consecutive G residues (G blocks). Alginates containing G blocks can be cross-linked by divalent cations and thereby form gels (35).Polysaccharide lyases (EC 4.2.2.-) are a group of enzymes which cleave the polymer chains via a β-elimination mechanism, resulting in the formation of a double bond at the newly formed nonreducing end. For alginate lyases, 4-deoxy-l-erythro-hex-4-enepyranosyluronate (denoted as Δ) is formed at the nonreducing end. Several such lyases have been purified from both alginate-producing and alginate-degrading organisms, as reviewed by Wong et al. (42). When they are classified according to primary structure, the alginate lyases belong to the polysaccharide-degrading enzyme families PL5, PL6, PL7, PL14, PL17, and PL18 (http://www.cazy.org). Alginate molecules may contain four different bonds (M-M, M-G, G-M, and G-G), and alginate lyases may therefore be classified according to their preferred substrate specificities. It is now possible to obtain pure mannuronan and nearly pure (MG)_n and G blocks (17, 19, 20), and this allows for an improved assessment of the substrate specificities of the alginate lyases.The following two alginate lyases have been characterized in A. vinelandii: the periplasmic AlgL that belongs to the PL5 family (15) and the extracellular bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7 (36, 37). AlgL is encoded by the alginate biosynthesis operon, similar to what has been found in all characterized alginate-producing bacteria. This enzyme cleaves M-M and M-G bonds (15), while AlgE7 preferably degrades G-MM and G-GM bonds (37). The latter enzyme is also able to introduce G residues in the alginate, thus creating the preferred substrate for the lyase.When A. vinelandii experiences a lack of nutrients, it will develop into a dormant cell designated cyst (30). The cell is then protected against desiccation by a multilayered coat, of which gel-forming alginate is a necessary part. Resuspension of cysts in a medium containing glucose leads to a germination process in which vegetative cells eventually escape from the cyst coat. It has been proposed that an alginate lyase may be involved in the rupture of the coat (43). AlgL is dispensable for germination (38), while the biological function of AlgE7 is unknown. In this report, we use the available draft genome sequence of A. vinelandii to identify three additional putative lyases and evaluate their and AlgE7''s role in growth, encystment, and germination of the bacterium.     

The Azotobacter vinelandii AlgE mannuronan C-5-epimerase family is essential for the in vivo control of alginate monomer composition and for functional cyst formation

The industrially widely used polysaccharide alginate is a co-polymer of β- d -mannuronic acid and α- l -guluronic acid (G), and the G residues originate from a polymer-level epimerization process catalysed by mannuronan C-5-epimerases. In the genome of the alginate-producing bacterium Azotobacter vinelandii genes encoding one periplasmic (AlgG) and seven secreted such epimerases (AlgE1–7) have been identified. Here we report the generation of a strain (MS163171) in which all the algE genes were inactivated by deletion ( algE1–4 and algE6–7 ) or interruption ( algE5 ). Shake flask-grown MS163171 produced a polymer containing less than 2% G ( algG still active), while wild-type alginates contained 25% G. Interestingly, addition of proteases to the MS163171 growth medium resulted in a strong increase in the chain lengths of the alginates produced. MS163171 was found to be unable to form functional cysts, which is a desiccation-resistant differentiated form developed by A. vinelandii under certain environmental conditions. We also generated mutants carrying interruptions in each separate algE gene, and a strain containing algE5 only. Studies of these mutants indicated that single algE gene inactivations, with the exception of algE3 , did not affect the fractional G content much. However, for all strains tested the alginate composition varied somewhat as a response to the growth conditions.     

A study of the reaction catalysed by alginate lyase VI from the sea mollusc, Littorina sp.

The molecular weight of polymeric alginic acid digested by alginate lyase (poly(1,4-beta-D-mannuronide) lyase, EC 4.2.2.3) was determined at various stages of the lysis. Low molecular weigh fragments were detected only after 60-100% lysis. Some high molecular weight fragments remained intact even after addition of a fresh aliquot of enzyme to the digest. The enzyme showed maximal activity at pH 5.6 in 0.05 M salt. Enzyme activity was stimulated by addition of 7.5 mM CaCl2 and 0.2 M NaCl, when the pH optimum was between 8 and 8.5. Only mannuronic acid was detected at the reducing end of fragments after exhausive enzymolysis, reduction and hydrolysis. On studying the reaction products by NMR, a double-bound signal (sigma = 5.98 ppm) was observed. A considerable decrease in intensity of the D-mannuronic acid residue signal was detected after hydrolysis of alginate lyase VI on poly-(ManUA-GulUA), but not poly(GulUA). The results suggest that alginate lyase VI may be an endoalginate lyase that splits glycoside bonds only between two mannuronic acid residues.     

Alginate lyase from Pseudomonas aeruginosa CF1/M1 prefers the hexameric oligomannuronate as substrate

In order to investigate the catalytic properties of alginate lyase from Pseudomonas aeruginosa CF1/M1, a clinical isolate, regarding the capability to perform β-elimination on oligomannuronates of defined length (2–9), the alginate lyase was purified from periplasmic extracts. A purification method for unsaturated and saturated oligomannuronates applying anionic exchange chromatography on a FPLC apparatus was established. The alginate lyase showed the highest activity, when hexamers were provided as substrate. This indicated that the alginate lyase best accommodates a chain of six alginate residues in the active center. As a minimum chain length, the pentameric oligomannuronate was still accepted as substrate. Mannuronate oligomers shorter than the pentamer were not accepted as substrate for alginate lyase. Furthermore, oligomer pattern analysis of polymannuronate which was subjected to β-elimination by alginate lyase revealed that the trimer is the most abundant oligomer. These data indicated that β-elimination and cleavage occurred at mannuronic acid residue no. 3 of the accommodated hexameric alginate chain.     

Mechanical properties of C-5 epimerized alginates

There is an increased need for alginate materials with both enhanced and controllable mechanical properties in the fields of food, pharmaceutical and specialty applications. In the present work, well-characterized algal polymers and mannuronan were enzymatically modified using C-5 epimerases converting mannuronic acid residues to guluronic acid in the polymer chain. Composition and sequential structure of controls and epimerized alginates were analyzed by (1)H NMR spectroscopy. Mechanical properties of Ca-alginate gels were further examined giving Young's modulus, syneresis, rupture strength, and elasticity of the gels. Both mechanical strength and elasticity of hydrogels could be improved and manipulated by epimerization. In particular, alternating sequences were found to play an important role for the final mechanical properties of alginate gels, and interestingly, a pure polyalternating sample resulted in gels with extremely high syneresis and rupture strength. In conclusion, enzymatic modification was shown to be a valuable tool in modifying the mechanical properties of alginates in a highly specific manner.     

Catalytic properties and specificity of a recombinant, overexpressed D-mannuronate lyase

Lysis of alginates and of their saturated and unsaturated fragments was monitored by 1H NMR spectroscopy. AlxM(B) alginate lyase performs beta-elimination on the mannuronic acid (M) residues. It does not cleave the guluronic acid (G) sequences, nor the M-G or the G-M diads. In consequence, it is a true mannuronate lyase. The end product of the reaction is O-(4-deoxy-alpha-L-ery-thro-hex-4-enopyranosyl-uronic acid)-(1->(4)-O-(beta-D-mannopyranosyluronic acid)-(1->4)-O-beta-D-mannpyranuronic acid. Viscosity measurements made during degradation of a polymannuronate alginate showed that AlxM(B) behaves as an endo-enzyme. HPLC analysis of the degradation products of oligomannuronates and oligoalginates suggested that the beta-elimination requires the interaction of the enzyme with at least three sequential mannuronic acid residues. The catalytic site may possess 5 sub-sites and accommodate pentamers with different M/G ratio. Kinetic measurements showed that the specificity constant Vm/Km increased with the number of mannuronic acid residues. AlxM(B) may be reversibly inhibited by heteropolymeric blocks in a competitive manner.     

Mannuronan C-5-epimerases and their application for in vitro and in vivo design of new alginates useful in biotechnology

The industrially important polysaccharide alginate is a linear copolymer of beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G). It is produced commercially by extraction from brown seaweeds, although some of the bacteria belonging to the genera Azotobacter and Pseudomonas also synthesize alginates. Alginates are synthesized as mannuronan, and varying amounts of the M residues in the polymer are then epimerized to G residues by mannuronan C-5-epimerases. The gel-forming, water-binding, and immunogenic properties of the polymer are dependent on the relative amount and sequence distribution of M and G residues. A family of seven calcium-dependent, secreted epimerases (AlgE1-7) from Azotobacter vinelandii have now been characterized, and in this paper the properties of all these enzymes are described. AlgE4 introduces alternating M and G residues into its substrate, while the remaining six enzymes introduce a mixture of continuous stretches of G residues and alternating sequences. Two of the enzymes, AlgE1 and AlgE3, are composed of two catalytically active domains, each introducing different G residue sequence patterns in alginate. These results indicate that the enzymes can be used for production of alginates with specialized properties.     

Identification and characterization of an Azotobacter vinelandii type I secretion system responsible for export of the AlgE-type mannuronan C-5-epimerases

Alginate is a linear copolymer of beta-d-mannuronic acid and its C-5-epimer, alpha-l-guluronic acid. During biosynthesis, the polymer is first made as mannuronan, and various fractions of the monomers are then epimerized to guluronic acid by mannuronan C-5-epimerases. The Azotobacter vinelandii genome encodes a family of seven extracellular such epimerases (AlgE1 to AlgE7) which display motifs characteristic for proteins secreted via a type I pathway. Putative ATPase-binding cassette regions from the genome draft sequence of the A. vinelandii OP strain and experimentally verified type I transporters from other species were compared. This analysis led to the identification of one putative A. vinelandii type I system (eexDEF). The corresponding genes were individually disrupted in A. vinelandii strain E, and Western blot analysis using polyclonal antibodies against all AlgE epimerases showed that these proteins were present in wild-type culture supernatants but absent from the eex mutant supernatants. Consistent with this, the wild-type strain and the eex mutants produced alginate with about 20% guluronic acid and almost pure mannuronan (< or =2% guluronic acid), respectively. The A. vinelandii wild type is able to enter a particular desiccation-tolerant resting stage designated cyst. At this stage, the cells are surrounded by a rigid coat in which alginate is a major constituent. Such a coat was formed by wild-type cells in a particular growth medium but was missing in the eex mutants. These mutants were also found to be unable to survive desiccation. The reason for this is probably that continuous stretches of guluronic acid residues are needed for alginate gel formation to take place.     

Acid gel formation in (pseudo) alginates with and without G blocks produced by epimerising mannuronan with C5 epimerases

The main scope of this paper is the characterization, in terms of viscoelastic and mechanical properties, of acid gels formed from solutions of mannuronan ALG (0%G/0%GG) and its enzymatically epimerised products. The epimerised products were obtained using recombinantly produced mannuronan C5 epimerases named AlgE1 and AlgE4, which catalyse the conversion of mannuronic residues into guluronic (G) and guluronic–mannuronic (GM) blocks, respectively. The products used in this study resulted from either the action of AlgE1 on mannuronan for 5 and 24 h (named ALG(44%G/32%GG) and ALG (68%G/59%GG), respectively) or AlgE4 on mannuronan (named ALG (47%G/0%GG)). d-gluconic acid-δ-lactone (GDL) was used as H⁺-donor to produce acidic gels. ALG (0%G/0%GG) yields strong, stable solid-like structures. As predicted by circular dichroism measurements performed at different pH, gelation of ALG (47%G/0%GG) occurs at lower values of pH (1) than those obtainable using GDL. Hydrochloric acid was therefore added to ALG (47%G/0%GG) solutions yielding rapid sol–gel transitions and gels with a remarkable resistance to thermal treatment.

The introduction of guluronic residues along the chain (ALG (44%G/32%GG)) causes a reduction in the storage modulus at the equilibrium with respect to that of ALG (0%G/0%GG) and the occurrence of negligible syneresis at the highest polymer concentrations. The increase in the average length of the G blocks (ALG (68%G/59%GG)) is accompanied by a further increase in the storage modulus without the occurrence of any significant syneresis.     

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

Pseudomonas aeruginosa is an opportunistic pathogen that forms chronic biofilm infections in the lungs of cystic fibrosis patients. A major component of the biofilm during these infections is the exopolysaccharide alginate, which is synthesized at the inner membrane as a homopolymer of 1–4-linked β-d-mannuronate. As the polymer passages through the periplasm, 22–44% of the mannuronate residues are converted to α-l-guluronate by the C5-epimerase AlgG to produce a polymer of alternating β-d-mannuronate and α-l-guluronate blocks and stretches of polymannuronate. To understand the molecular basis of alginate epimerization, the structure of Pseudomonas syringae AlgG has been determined at 2.1-Å resolution, and the protein was functionally characterized. The structure reveals that AlgG is a long right-handed parallel β-helix with an elaborate lid structure. Functional analysis of AlgG mutants suggests that His³¹⁹ acts as the catalytic base and that Arg³⁴⁵ neutralizes the acidic group during the epimerase reaction. Water is the likely catalytic acid. Electrostatic surface potential and residue conservation analyses in conjunction with activity and substrate docking studies suggest that a conserved electropositive groove facilitates polymannuronate binding and contains at least nine substrate binding subsites. These subsites likely align the polymer in the correct register for catalysis to occur. The presence of multiple subsites, the electropositive groove, and the non-random distribution of guluronate in the alginate polymer suggest that AlgG is a processive enzyme. Moreover, comparison of AlgG and the extracellular alginate epimerase AlgE4 of Azotobacter vinelandii provides a structural rationale for the differences in their Ca²⁺ dependence.     

Mode of action and subsite studies of the guluronan block-forming mannuronan C-5 epimerases AlgE1 and AlgE6

AlgE1, AlgE5 and AlgE6 are members of a family of mannuronan C-5 epimerases encoded by the bacterium Azotobacter vinelandii, and are active in the biosynthesis of alginate, where they catalyse the post-polymerization conversion of beta-D-mannuronic acid (M) residues into alpha-L-guluronic acid residues (G). All enzymes show preference for introducing G-residues neighbouring a pre-existing G. They also have the capacity to convert single M residues flanked by G, thus 'condensing' G-blocks to form almost homopolymeric guluronan. Analysis of the length and distribution of G-blocks based on specific enzyme degradation combined with size-exclusion chromatography, electrospray ionization MS, HPAEC-PAD (high-performance anion-exchange chromatography and pulsed amperometric detection), MALDI (matrix-assisted laser-desorption ionization)-MS and NMR revealed large differences in block length and distribution generated by AlgE1 and AlgE6, probably reflecting their different degree of processivity. When acting on polyMG as substrates, AlgE1 initially forms only long homopolymeric G-blocks >50, while AlgE6 gives shorter blocks with a broader block size distribution. Analyses of the AlgE1 and AlgE6 subsite specificities by the same methodology showed that a mannuronan octamer and heptamer respectively were the minimum substrate chain lengths needed to accommodate enzyme activities. The fourth M residue from the non-reducing end is epimerized first by both enzymes. When acting on MG-oligomers, AlgE1 needed a decamer while AlgE6 an octamer to accommodate activity. By performing FIA (flow injection analysis)-MS on the lyase digests of epimerized and standard MG-oligomers, the M residue in position 5 from the non-reducing end was preferentially attacked by both enzymes, creating an MGMGGG-sequence (underlined and boldface indicate the epimerized residue).     

Role of the Pseudomonas fluorescens alginate lyase (AlgL) in clearing the periplasm of alginates not exported to the extracellular environment

Alginate is an industrially widely used polysaccharide produced by brown seaweeds and as an exopolysaccharide by bacteria belonging to the genera Pseudomonas and Azotobacter. The polymer is composed of the two sugar monomers mannuronic acid and guluronic acid (G), and in all these bacteria the genes encoding 12 of the proteins essential for synthesis of the polymer are clustered in the genome. Interestingly, 1 of the 12 proteins is an alginate lyase (AlgL), which is able to degrade the polymer down to short oligouronides. The reason why this lyase is associated with the biosynthetic complex is not clear, but in this paper we show that the complete lack of AlgL activity in Pseudomonas fluorescens in the presence of high levels of alginate synthesis is toxic to the cells. This toxicity increased with the level of alginate synthesis. Furthermore, alginate synthesis became reduced in the absence of AlgL, and the polymers contained much less G residues than in the wild-type polymer. To explain these results and other data previously reported in the literature, we propose that the main biological function of AlgL is to degrade alginates that fail to become exported out of the cell and thereby become stranded in the periplasmic space. At high levels of alginate synthesis in the absence of AlgL, such stranded polymers may accumulate in the periplasm to such an extent that the integrity of the cell is lost, leading to the observed toxic effects.     

Time-resolved 1H and 13C NMR spectroscopy for detailed analyses of the Azotobacter vinelandii mannuronan C-5 epimerase reaction

AlgE2, AlgE4, and AlgE6 are members of a family of mannuronan C-5 epimerases encoded by Azotobacter vinelandii, and are active in the biosynthesis of alginate, where they catalyze the post-polymerization conversion of beta-D-mannuronic acid residues into alpha-L-guluronic acid residues. To study the kinetics and mode of action of these enzymes, homopolymeric mannuronan and other alginate samples with various composition were epimerized by letting the enzymatic reaction take place in an NMR tube. Series of 1H NMR spectra were recorded to obtain a time-resolved picture of the epimerization progress and the formation of specific monomer sequences. Starting from mannuronan, guluronic acid contents of up to 82% were introduced by the enzymes, and the product specificity, substrate selectivity, and reaction rates have been investigated. To obtain direct information of the GulA-block formation, similar experiments were performed using a 13C-1-enriched mannuronan as substrate. The NMR results were found to be in good agreement with data obtained by a radioisotope assay based on 3H-5-labeled substrates.     

Mode of action of recombinant Azotobacter vinelandii mannuronan C-5 epimerases AlgE2 and AlgE4.

The enzymes mannuronan C-5 epimerases catalyze conversion of beta-D-mannuronic acid to alpha-L-guluronic acid in alginates at the polymer level and thereby introduce sequences that have functional properties relevant to gelation. The enzymatic conversion by recombinant mannuronan C-5 epimerases AlgE4 and AlgE2 on alginate type substrates with different degree of polymerization and initial low fraction of alpha-L-guluronic acid was investigated. Essentially no enzymatic activity was found for fractionated mannuronan oligomer substrates with an average degree of polymerization, DP(n), less than or equal 6, whereas increasing the DP(n) yielded increased epimerization activity. This indicates that these enzymes have an active site consisting of binding domains for consecutive residues that requires interaction with 7 or more consecutive residues to show enzymatic activity. The experimentally determined kinetics of the reaction, and the residue sequence arrangement introduced by the epimerization, were modeled using Monte Carlo simulation accounting for the various competing intrachain substrates and assuming either a processive mode of action or preferred attack. The comparison between experimental data and simulation results suggests that epimerization by AlgE4 is best described by a processive mode of action, whereas the mode of action of AlgE2 appears to be more difficult to determine.     

The recombinant Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 epimerizes alginate by a nonrandom attack mechanism

The Ca2+-dependent mannuronan C-5-epimerase AlgE4 is a representative of a family of Azotobacter vinelandii enzymes catalyzing the polymer level epimerization of beta-D-mannuronic acid (M) to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate. The reaction product of recombinantly produced AlgE4 is predominantly characterized by an alternating sequence distribution of the M and G residues (MG blocks). AlgE4 was purified after intracellular overexpression in Escherichia coli, and the activity was shown to be optimal at pH values between 6.5 and 7.0, in the presence of 1-3 mM Ca2+, and at temperatures near 37 degrees C. Sr2+ was found to substitute reasonably well for Ca2+ in activation, whereas Zn2+ strongly inhibited the activity. During epimerization of alginate, the fraction of GMG blocks increased linearly as a function of the total fraction of G residues and comparably much faster than that of MMG blocks. These experimental data could not be accounted for by a random attack mechanism, suggesting that the enzyme either slides along the alginate chain during catalysis or recognizes a pre-existing G residue as a preferred substrate in its consecutive attacks.     

Characterization of AlgMsp,an Alginate Lyase from Microbulbifer sp. 6532A

Alginate is a polysaccharide produced by certain seaweeds and bacteria that consists of mannuronic acid and guluronic acid residues. Seaweed alginate is used in food and industrial chemical processes, while the biosynthesis of bacterial alginate is associated with pathogenic Pseudomonas aeruginosa. Alginate lyases cleave this polysaccharide into short oligo-uronates and thus have the potential to be utilized for both industrial and medicinal applications. An alginate lyase gene, algMsp, from Microbulbifer sp. 6532A, was synthesized as an E.coli codon-optimized clone. The resulting 37 kDa recombinant protein, AlgMsp, was expressed, purified and characterized. The alginate lyase displayed highest activity at pH 8 and 0.2 M NaCl. Activity of the alginate lyase was greatest at 50°C; however the enzyme was not stable over time when incubated at 50°C. The alginate lyase was still highly active at 25°C and displayed little or no loss of activity after 24 hours at 25°C. The activity of AlgMsp was not dependent on the presence of divalent cations. Comparing activity of the lyase against polymannuronic acid and polyguluronic acid substrates showed a higher turnover rate for polymannuronic acid. However, AlgMSP exhibited greater catalytic efficiency with the polyguluronic acid substrate. Prolonged AlgMsp-mediated degradation of alginate produced dimer, trimer, tetramer, and pentamer oligo-uronates.     

Evidence that the algI/algJ gene cassette, required for O acetylation of Pseudomonas aeruginosa alginate, evolved by lateral gene transfer

Pseudomonas aeruginosa strains, isolated from chronically infected patients with cystic fibrosis, produce the O-acetylated extracellular polysaccharide, alginate, giving these strains a mucoid phenotype. O acetylation of alginate plays an important role in the ability of mucoid P. aeruginosa to form biofilms and to resist complement-mediated phagocytosis. The O-acetylation process is complex, requiring a protein with seven transmembrane domains (AlgI), a type II membrane protein (AlgJ), and a periplasmic protein (AlgF). The cellular localization of these proteins suggests a model wherein alginate is modified at the polymer level after the transport of O-acetyl groups to the periplasm. Here, we demonstrate that this mechanism for polysaccharide esterification may be common among bacteria, since AlgI homologs linked to type II membrane proteins are found in a variety of gram-positive and gram-negative bacteria. In some cases, genes for these homologs have been incorporated into polysaccharide biosynthetic operons other than for alginate biosynthesis. The phylogenies of AlgI do not correlate with the phylogeny of the host bacteria, based on 16S rRNA analysis. The algI homologs and the gene for their adjacent type II membrane protein present a mosaic pattern of gene arrangement, suggesting that individual components of the multigene cassette, as well as the entire cassette, evolved by lateral gene transfer. AlgJ and the other type II membrane proteins, although more diverged than AlgI, contain conserved motifs, including a motif surrounding a highly conserved histidine residue, which is required for alginate O-acetylation activity by AlgJ. The AlgI homologs also contain an ordered series of motifs that included conserved amino acid residues in the cytoplasmic domain CD-4; the transmembrane domains TM-C, TM-D, and TM-E; and the periplasmic domain PD-3. Site-directed mutagenesis studies were used to identify amino acids important for alginate O-acetylation activity, including those likely required for (i) the interaction of AlgI with the O-acetyl precursor in the cytoplasm, (ii) the export of the O-acetyl group across the cytoplasmic membrane, and (iii) the transfer of the O-acetyl group to a periplasmic protein or to alginate. These results indicate that AlgI belongs to a family of membrane proteins required for modification of polysaccharides and that a mechanism requiring an AlgI homolog and a type II membrane protein has evolved by lateral gene transfer for the esterification of many bacterial extracellular polysaccharides.