Expression, purification, crystallization and preliminary X-ray analysis of<i>Pseudomonas aeruginosa</i>AlgL

Wolfram, F.; Arora, Kritica; Robinson, Howard; Neculai, Ana Mirela; Yip, P.; Howell, P. Lynne

doi:10.1107/s1744309112012808

Cited by 11 publications

(18 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Higher yields of AlgL were obtained using Co 2+ -NTA columns instead of Ni 2+ -NTA, which was used in previous work (9) (data not shown). Samples supplemented with 10% (v/v) glycerol and stored at −80 °C were stable for several months.…”

Section: Resultsmentioning

confidence: 70%

Functional Characterization of AlgL, an Alginate Lyase from Pseudomonas aeruginosa

Farrell

Tipton

2012

Biochemistry

View full text Add to dashboard Cite

Alginate lyase (AlgL) catalyzes the cleavage of the polysaccharide alginate through a β-elimination reaction. In Pseudomonas aeruginosa algL is part of the alginate biosynthetic operon, and although it is required for alginate biosynthesis, it is not clear why. Steady-state kinetic studies were performed to characterize its substrate specificity, and revealed that AlgL operates preferentially on non-acetylated alginate or its precursor mannuronan. Mature alginate is secreted as a partially acetylated polysaccharide, so this observation is consistent with suggestions that AlgL serves to degrade mislocalized alginate that is trapped in the periplasmic space. The kcat/Km for the reaction increased linearly with the number of residues in the substrate, from 2.1×105 M−1s−1 for substrate containing 16 residues to 7.9×106 M−1s−1 for substrate with 280 residues. Over the same substrate size range, kcat varied between 10 s−1 and 30 s−1. The variation in kcat/Km with substrate length suggests that AlgL operates in a processive manner. AlgL displayed a surprising lack of stereospecificity, in that it was able to catalyze cleavage adjacent to either mannuronate or guluronate residues in alginate. Thus, the enzyme is able to remove the C5 proton from both mannuronate and guluronate, which are C5 epimers. Exhaustive digestion of alginate by AlgL generated dimeric and trimeric products, which were characterized by 1H NMR spectroscopy and mass spectrometry. Rapid-mixing chemical quench studies revealed that there was no lag in dimer or trimer production, indicating that AlgL operates as an exopolysaccharide lyase.

show abstract

Section: Resultsmentioning

confidence: 70%

Functional Characterization of AlgL, an Alginate Lyase from Pseudomonas aeruginosa

Farrell

Tipton

2012

Biochemistry

View full text Add to dashboard Cite

show abstract

“…Because of its limited solubility, cellohexaose was tested at 3 mM, the maximum that our assay allowed. We observed a significant increase in the rate of formation of paranitrophenolate in the presence of the tetrasaccharide, pentasaccharide and hexasaccharide (16.8 +/-0.5, 31.6 +/-1.5, and 33.1 +/-1.1 nmol min -1 mg protein -1 , respectively; ANOVA, F (6,20) = 1283, p < 0.0001; Fig. 2).…”

Section: Resultsmentioning

confidence: 81%

“…Polymerization occurs concomitantly with transport across the inner membrane through a pore in BcsA that leads to emergence of the growing chain into the periplasm where it interacts with BcsB, a protein containing a carbohydrate-binding domain (18). While the precise molecular mechanism of cellulose export across the periplasm and outer membrane of bacteria is still unknown, in the analogous alginate system this process occurs through the coordinated action of a tetratricopeptide repeat (TPR)containing protein (AlgK) and an outer membrane β-barrel containing protein (AlgE), with release of the nascent chain occurring through the action of alginate lyase (AlgL) (15,16,20,21). BcsC in the cellulose system has been proposed to carry out the functions of AlgK and AlgE, since it has homologous regions for both the TPR and β-barrel domains (15,21).…”

mentioning

confidence: 99%

“…BcsC in the cellulose system has been proposed to carry out the functions of AlgK and AlgE, since it has homologous regions for both the TPR and β-barrel domains (15,21). Studies with recombinant BcsZ have further provided evidence for this model, as functional and structural results demonstrated that BcsZ hydrolyzes cellulose polymers and may reside in the periplasm; thereby positioning it to act in a similar manner to AlgL (20,22).…”

mentioning

confidence: 99%

See 1 more Smart Citation

The Escherichia coli cellulose synthase subunit G (BcsG) is a Zn2+-dependent phosphoethanolamine transferase

Anderson

Burnett

Hiscock

et al. 2020

Journal of Biological Chemistry

View full text Add to dashboard Cite

Bacterial biofilms are cellular communities that produce an adherent matrix. Exopolysaccharides are key structural components of this matrix and are required for the assembly and architecture of biofilms produced by a wide variety of microorganisms. The human bacterial pathogens Escherichia coli and Salmonella enterica produce a biofilm matrix composed primarily of the exopolysaccharide phosphoethanolamine (pEtN) cellulose. Once thought to be composed of only underivatized cellulose, the pEtN modification present in these matrices has been implicated in the overall architecture and integrity of the biofilm. However, an understanding of the mechanism underlying pEtN derivatization of the cellulose exopolysaccharide remains elusive. The bacterial cellulose synthase subunit G (BcsG) is a predicted inner membrane–localized metalloenzyme that has been proposed to catalyze the transfer of the pEtN group from membrane phospholipids to cellulose. Here we present evidence that the C-terminal domain of BcsG from E. coli (EcBcsGΔN) functions as a phosphoethanolamine transferase in vitro with substrate preference for cellulosic materials. Structural characterization of EcBcsGΔN revealed that it belongs to the alkaline phosphatase superfamily, contains a Zn2+ ion at its active center, and is structurally similar to characterized enzymes that confer colistin resistance in Gram-negative bacteria. Informed by our structural studies, we present a functional complementation experiment in E. coli AR3110, indicating that the activity of the BcsG C-terminal domain is essential for integrity of the pellicular biofilm. Furthermore, our results established a similar but distinct active-site architecture and catalytic mechanism shared between BcsG and the colistin resistance enzymes.

show abstract

“…The proposed function of AlgK was derived, in part, from phenotypic studies demonstrating that in the absence of AlgK, alginate is degraded by AlgL [28,57,58]. The presence of TPRs in the structure of AlgK suggests that it may serve as a scaffold to which the other periplasmic Alg proteins interact to form a multiprotein complex (Figure 3) [29].…”

Section: Synthase-dependent Production Of Alginatementioning

confidence: 99%

Synthase-dependent exopolysaccharide secretion in Gram-negative bacteria

Whitney

Howell

2013

Trends in Microbiology

221

231

View full text Add to dashboard Cite

The biosynthesis and export of bacterial cell-surface polysaccharides is known to occur through several distinct mechanisms. Recent advances in the biochemistry and structural biology of several proteins in synthase-dependent polysaccharide secretion systems have identified key conserved components of this pathway in Gram-negative bacteria. These components include an innermembrane-embedded polysaccharide synthase, a periplasmic tetratricopeptide repeat (TPR)-containing scaffold protein, and an outer-membrane β-barrel porin. There is also increasing evidence that many synthase-dependent systems are post-translationally regulated by the bacterial second messenger bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). Here, we compare these core proteins in the context of the alginate, cellulose, and poly-β-D-Nacetylglucosamine (PNAG) secretion systems.

show abstract

Expression, purification, crystallization and preliminary X-ray analysis ofPseudomonas aeruginosaAlgL

Cited by 11 publications

References 22 publications

Functional Characterization of AlgL, an Alginate Lyase from Pseudomonas aeruginosa

Functional Characterization of AlgL, an Alginate Lyase from Pseudomonas aeruginosa

The Escherichia coli cellulose synthase subunit G (BcsG) is a Zn2+-dependent phosphoethanolamine transferase

Synthase-dependent exopolysaccharide secretion in Gram-negative bacteria

Contact Info

Product

Resources

About