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Investigation of the substrate specificity of K5 lyase A from K5A bacteriophage.

ABSTRACT: K5 lyase A (KflA) is a tailspike protein from the K5A phage that catalyzes the degradation of the capsule polysaccharide of K5 strains of Escherichia coli. The K5 E. coli capsule polysaccharide, also known as heparosan, is composed of the disaccharide repeating unit of [-4)-GlcA-?(1,4)-GlcNAc-?(1-] and therefore identical to the biological precursor of heparin and heparan sulfate (HS). KflA could supplement the heparin lyases for heparin and HS analysis. The first part of this study aimed to clarify ambiguity resulting from the revision of the KflA amino acid sequence in 2010 from that published in 2000. We found that only the expression of the updated sequence gave a soluble active enzyme, which produced heparosan degradation products similar to those of previous studies. Next, we examined the specificity of KflA toward heparosan oligosaccharides of varying sizes, all containing a single N-sulfated glucosamine (GlcNS) residue. The presence of GlcNS in an octasaccharide and a nonasaccharide chain directed cleavage by KflA to a single position at the reducing end of the substrate. However, an N-sulfated decasaccharide exhibited extensive cleavage at the nonreducing end of the chain, illustrating a distinct change in the cleavage pattern of KflA toward substrates of differing sizes. Because KflA is able to cleave a substrate containing isolated GlcNS residues, this enzyme could be used for the analysis of low-sulfate content HS domains.

SUBMITTER: O'Leary TR

PROVIDER: S-EPMC3505012 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Investigation of the substrate specificity of K5 lyase A from K5A bacteriophage.

O'Leary Timothy R TR Xu Yongmei Y Liu Jian J

Glycobiology 20120926 1

K5 lyase A (KflA) is a tailspike protein from the K5A phage that catalyzes the degradation of the capsule polysaccharide of K5 strains of Escherichia coli. The K5 E. coli capsule polysaccharide, also known as heparosan, is composed of the disaccharide repeating unit of [-4)-GlcA-β(1,4)-GlcNAc-α(1-] and therefore identical to the biological precursor of heparin and heparan sulfate (HS). KflA could supplement the heparin lyases for heparin and HS analysis. The first part of this study aimed to cla ...[more]

PMID: 23019155

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Project description:The Escherichia coli K5 capsular polysaccharide [-4)-betaGlcA-(1, 4)-alphaGlcNAc-(1-] is a receptor for the capsule-specific bacteriophage K5A. Associated with the structure of bacteriophage K5A is a polysaccharide lyase which degrades the K5 capsule to expose the underlying bacterial cell surface. The bacteriophage K5A lyase gene (kflA) was cloned and sequenced. The kflA gene encodes a polypeptide with a predicted molecular mass of 66.9 kDa and which exhibits amino acid homology with ElmA, a K5 polysaccharide lyase encoded on the chromosome of E. coli SEBR 3282. There was only limited nucleotide homology between the kflA and elmA genes, suggesting that these two genes are distinct and either have been derived from separate progenitors or have diverged from a common progenitor for a considerable length of time. Southern blot analysis revealed that kflA was not present on the chromosome of the E. coli strains examined. In contrast, elmA was present in a subset of E. coli strains. Homology was observed between DNA flanking the kflA gene of bacteriophage K5A and DNA flanking a small open reading frame (ORF(L)) located 5' of the endosialidase gene of the E. coli K1 capsule-specific bacteriophage K1E. The DNA homology between these noncoding sequences indicated that bacteriophages K5A and K1E were related. The deduced polypeptide sequence of ORF(L) in bacteriophage K1E exhibited homology to the N terminus of KflA from bacteriophage K5A, suggesting that ORF(L) is a truncated remnant of KflA. The presence of this truncated kflA gene implies that bacteriophage K1E has evolved from bacteriophage K5A by acquisition of the endosialidase gene and subsequent loss of functional kflA. A (His)(6)-KflA fusion protein was overexpressed in E. coli and purified to homogeneity with a yield of 4.8 mg per liter of bacterial culture. The recombinant enzyme was active over a broad pH range and NaCl concentration and was capable of degrading K5 polysaccharide into a low-molecular-weight product.

Project description:Anionic polysaccharides are of growing interest in the biotechnology industry due to their potential pharmaceutical applications in drug delivery and wound treatment. Chemical composition and polymer length strongly influence the physical and biological properties of the polysaccharide and thus its potential industrial and medical applications. One promising approach to determining monomer composition and controlling the degree of polymerization involves the use of polysaccharide lyases, which catalyze the depolymerization of anionic polysaccharides via a β-elimination mechanism. Utilization of these enzymes for the production of custom-made oligosaccharides requires a high degree of control over substrate specificity. Previously, we characterized a polysaccharide lyase (Smlt1473) from Stenotrophomonas maltophilia k279a, which exhibited significant activity against hyaluronan (HA), poly-β-d-glucuronic acid (poly-GlcUA), and poly-β-d-mannuronic acid (poly-ManA) in a pH-regulated manner. Here, we utilize a sequence structure guided approach based on a homology model of Smlt1473 to identify nine putative substrate-binding residues and examine their effect on substrate specificity via site-directed mutagenesis. Interestingly, single point mutations H221F and R312L resulted in increased activity and specificity toward poly-ManA and poly-GlcUA, respectively. Furthermore, a W171A mutant nearly eliminated HA activity, while increasing poly-ManA and poly-GlcUA activity by at least 35%. The effect of these mutations was analyzed by comparison with the high resolution structure of Sphingomonas sp. A1-III alginate lyase in complex with poly-ManA tetrasaccharide and by taking into account the structural differences between HA, poly-GlcUA, and poly-ManA. Overall, our results demonstrate that even minor changes in active site architecture have a significant effect on the substrate specificity of Smlt1473, whose structural plasticity could be applied to the design of highly active and specific polysaccharide lyases.

Dataset Information

Investigation of the substrate specificity of K5 lyase A from K5A bacteriophage.

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Investigation of the substrate specificity of K5 lyase A from K5A bacteriophage.

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