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 \textgreater14 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.
%0 Journal Article
%1 bergfeld_biochemical_2007
%A Bergfeld, Anne K
%A Claus, Heike
%A Vogel, Ulrich
%A MĂźhlenhoff, Martina
%D 2007
%J The Journal of Biological Chemistry
%K Acetyltransferases, Base Chain Cloning, Data, Deletion, Escherichia Kinetics, Molecular Molecular, Mutagenesis, Plasmids, Polymerase Proteins, Reaction, Recombinant Sequence Sequence, Specificity Substrate bacterial_capsules coli coli, {Site-Directed},
%N 30
%P 22217--22227
%R 10.1074/jbc.M703044200
%T Biochemical characterization of the polysialic acid-specific O-acetyltransferase NeuO of Escherichia coli K1
%U http://www.ncbi.nlm.nih.gov/pubmed/17519228
%V 282
%X 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 \textgreater14 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.
@article{bergfeld_biochemical_2007,
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 {\textgreater}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.},
added-at = {2011-06-24T11:21:47.000+0200},
author = {Bergfeld, Anne K and Claus, Heike and Vogel, Ulrich and MĂźhlenhoff, Martina},
biburl = {https://www.bibsonomy.org/bibtex/271247123c7e4a07cf46d6530ea8b1fdc/ag_vogel},
doi = {10.1074/jbc.M703044200},
interhash = {4459f6e8cac846067cf6873ab0570132},
intrahash = {71247123c7e4a07cf46d6530ea8b1fdc},
issn = {0021-9258},
journal = {The Journal of Biological Chemistry},
keywords = {Acetyltransferases, Base Chain Cloning, Data, Deletion, Escherichia Kinetics, Molecular Molecular, Mutagenesis, Plasmids, Polymerase Proteins, Reaction, Recombinant Sequence Sequence, Specificity Substrate bacterial_capsules coli coli, {Site-Directed},},
month = jul,
note = {{PMID:} 17519228},
number = 30,
pages = {22217--22227},
timestamp = {2011-08-08T15:46:42.000+0200},
title = {Biochemical characterization of the polysialic acid-specific O-acetyltransferase {NeuO} of Escherichia coli K1},
url = {http://www.ncbi.nlm.nih.gov/pubmed/17519228},
volume = 282,
year = 2007
}