HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 282, Issue 30, 22217-22227, July 27, 2007

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 282/30/22217    most recent M703044200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bergfeld, A. K. Articles by Mühlenhoff, M. Search for Related Content PubMed PubMed Citation Articles by Bergfeld, A. K. Articles by Mühlenhoff, M. Social Bookmarking                      What's this?

Biochemical Characterization of Thepolysialic Acid-specific O-Acetyltransferase NeuO of Escherichia coli K1^*

Anne K. Bergfeld, Heike Claus, Ulrich Vogel, and Martina Mühlenhoff¹

From the Department of Cellular Chemistry, Medical School Hannover, Carl-Neuberg-Strasse 1, Hannover 30625 and the Institute for Hygiene and Microbiology, University of Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany

Escherichia coli K1 is a leading pathogen in neonatal sepsis and meningitis. The K1 capsule, composed of 2,8-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 -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 -helix family are combined with a unique regulatory mechanism based on variable N-terminal protein extensions formed by tandem copies of an RLKTQDS heptad.

Received for publication, April 11, 2007 , and in revised form, May 14, 2007.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AJ783705.

^* This work was supported by grants of the Deutsche Forschungsgemeinschaft (Grant VO 718/4-1 to U. V. and Grant MU 1774/2-1 to M. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3.

¹ To whom correspondence should be addressed: Tel.: 49-511-532-9807; Fax: 49-511-532-3956; E-mail: muehlenhoff.martina{at}mh-hannover.de.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				A. K. Bergfeld, H. Claus, N. K. Lorenzen, F. Spielmann, U. Vogel, and M. Muhlenhoff The Polysialic Acid-specific O-Acetyltransferase OatC from Neisseria meningitidis Serogroup C Evolved Apart from Other Bacterial Sialate O-Acetyltransferases J. Biol. Chem., January 2, 2009; 284(1): 6 - 16. [Abstract] [Full Text] [PDF]