%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 }