Abstract
Escherichia coli K1 is a leading pathogen in neonatal sepsis and meningitis. The K1 capsule, composed of alpha2,8-linked polysialic acid, represents the major virulence factor. In some K1 strains, phase-variable O-acetylation of the capsular polysaccharide is observed, a modification that is catalyzed by the prophage-encoded O-acetyltransferase NeuO. Phase variation is mediated by changes in the number of heptanucleotide repeats within the 5'-coding region of neuO, and full-length translation is restricted to repeat numbers that are a multiple of three. To understand the biochemical basis of K1 capsule O-acetylation, NeuO encoded by alleles containing 0, 12, 24, and 36 repeats was expressed and purified to homogeneity via a C-terminal hexahistidine tag. All NeuO variants assembled into hexamers and were enzymatically active with a high substrate specificity toward polysialic acid with >14 residues. Remarkably, the catalytic efficiency (k(cat)/K(m)(donor)) increased linearly with increasing numbers of repeats, revealing a new mechanism for modulating NeuO activity. Using homology modeling, we predicted a three-dimensional structure primarily composed of a left-handed parallel beta-helix with one protruding loop. Two amino acids critical for catalytic activity were identified and corresponding alanine substitutions, H119A and W143A, resulted in a complete loss of activity without affecting the oligomerization state. Our results indicate that in NeuO typical features of an acetyltransferase of the left-handed beta-helix family are combined with a unique regulatory mechanism based on variable N-terminal protein extensions formed by tandem copies of an RLKTQDS heptad.
Highlights
Escherichia coli K1 (E. coli K1)2 is one of the main organisms causing bacterial sepsis and meningitis during the neonatal period [1,2,3]
Expression levels and protein yields increased with increasing numbers of variable number of tandem repeats (VNTRs), suggesting that heptads contribute to protein stability
We succeeded for the first time in purification and characterization of a polysialic acid (polySia) specific O-acetyltransferase
Summary
BamHI and XhoI sites (underlined) in forward and reverse primers, respectively, were used for subcloning of the PCR products into the. BamHI/XhoI fragments of the corresponding PCR products were either subcloned in pET22b-Strep for recombinant expression in E. coli BL21(DE3). For comparison of NeuO activity towards colominic acid, CMP-Neu5Ac, Neu5Ac, and Neu5Ac oligomers of different length the concentration of all acceptor substrates were normalized to equal sialic acid content (6.85 mM) and thereby to an equal number of acceptor sites. The above described pET-based plasmids containing neuO encoding either wild-type or mutated variants with the amino acid exchanges H119A and W143A were used as a template. 1 mg of affinity purified wild-type or mutant NeuO was applied to the column and eluted protein was monitored by absorbance at 280 nm The column was equilibrated with 100 mM Tris-HCl pH 8.0/ 150 mM NaCl and calibrated with molecular weight standards (Sigma) thyroglobulin (669 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome C (12.4 kDa). 1 mg of affinity purified wild-type or mutant NeuO was applied to the column and eluted protein was monitored by absorbance at 280 nm
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