Expression, mutagenesis and kinetic analysis of recombinant K1E endosialidase to define the site of proteolytic processing and requirements for catalysis

Abstract

Catalytically active, recombinant fusion proteins of bacteriophage E endosialidase were expressed and purified from Escherichia coli. Constructs with different fusion partners added to the amino terminus of the endosialidase were enzymatically active. A post-translational proteolytic cleavage was shown to occur between serine 706 and aspartate 707 to generate the 76 kDa mature enzyme from the 90 kDa translation product. Endosialidase truncated at the C-terminus from aspartate 707 was observed to have the same 76 kDa molecular weight as wild-type enzyme using denaturing SDS-PAGE but, under native PAGE conditions, was not observed to form the approximately 250 kDa trimeric wild-type enzyme, implying that the C-terminus of the enzyme may be required for correct assembly of active trimer, rather than as part of the active site as has been previously suggested. Mutagenesis of aspartate 138 to alanine greatly reduced enzyme activity whereas conversion of other selected aspartate residues to alanine had less effect, consistent with similarities between the structure and cata-lytic mechanism of bacteriophage E endosialidase and those of exosialidases.

A tale of tails: Sialidase is key to success in a model of phage therapy against K1-capsulated Escherichia coli

“…Both K1-dep phage tailspike genes contain an endosialidase, with greater than 96% amino acid sequence identity to the enzyme characterized from the tail of phage 63D (Kataoka et al, 2006; Machida et al, 2000a; Machida et al, 2000b). Both K1-dep(2) and K1-dep(3) (K1E and K1–5, respectively) also have documented endosialidases (Leggate et al, 2002; Leiman et al, 2007; Muhlenhoff et al, 2003). In contrast, sequences of the three K1-ind phages indicate a glycosidase that is most closely related to the coliphage HK620 tailspike.…”

Prevention and Cure of Systemic Escherichia coli K1 Infection by Modification of the Bacterial Phenotype

“…The endoE gene open reading frame was amplified by PCR with cloned full-length DNA as a template and ligated into plasmid pQE30 (Qiagen, Ltd., Crawley, United Kingdom) by standard cloning technology to introduce a histidine tag at the N terminus of the recombinant protein; since we have found that the translated gene product undergoes proteolytic cleavage at a site close to the C terminus (15), the location of this tag is critical for subsequent purification of the fusion product. The plasmid was introduced into E. coli BL21(DE3), and the vector was grown in 2xTY broth (16 g of Bacto tryptone, 10 g of yeast extract, and 5 g of NaCl per liter) in controlled batch fermentation culture by using an Electrolab 300 Series Microprocessor-steered 10-liter fermentor with IPTG (isopropyl-␤-D-thiogalactopyranoside) at optical density at 600 nm (OD 600 ) of 0.3 for up to 6 h. Recombinant protein was purified by using immobilized metal (Ni) affinity chromatography of freeze-thawed sonicated bacterial lysates to yield up to 6 mg of His 6 -tagged recombinant endoE/ liter.…”

“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting indicated a very high purity of product, with no proteins detectable other than the 76-kDa recombinant endoE fusion product. Batches of His 6 -tagged recombinant enzyme were shown to be catalytically active by using the thiobarbiturate assay for release of N-acetylneuraminic acid from K1 polysaccharide extracted from E. coli LP1674 (15,17). There was no loss of enzyme function due to storage of solutions at 4°C for periods of up to 1 year.…”

“…Compromising the expression of PSA at the surface of E. coli K1 strains during the course of infection would allow mediators of the host's defenses to interact with underlying structures and eliminate the bacteria. We have identified, characterized, cloned, sequenced, and expressed in high yield in bacteria (15,17,29) an enzyme, endosialidase E (endoE), carried by a bacteriophage infecting E. coli K1 strains, that has an absolute specificity for ␣-2,8 sialic acid linkages and rapidly hydrolyzes the E. coli K1 capsular polysaccharide (29). We investigated the capacity of endoE to modulate the virulence of pathogenic E. coli K1 strains in vivo and in vitro.…”

Endosialidases: Versatile Tools for the Study of Polysialic Acid

“…Originally endosialidase purification has involved ammonium sulfate precipitation as well as hydrophobic, gel filtration, and DEAE-ion-exchange chromatography [101,104]. More conveniently, endosialidase is produced as a recombinant enzyme as His6-tagged, GST-tagged, and MBP-tagged fusion proteins [14,71,154].…”