The polysialic acid-specific O-acetyltransferase OatC from Neisseria meningitidis serogroup C evolved apart from other bacterial sialate O-acetyltransferases.
Neisseria meningitidis serogroup C is a major cause of bacterial meningitis and septicaemia. This human pathogen is protected by a capsule composed of alpha2,9-linked polysialic acid that represents an important virulence factor. In the majority of strains, the capsular polysaccharide is modified by O-acetylation at C-7 or C-8 of the sialic acid residues. The gene encoding the capsule modifying O-acetyltransferase is part of the capsule gene complex and shares no sequence similarities with other proteins. Here, we describe the purification and biochemical characterization of recombinant OatC. The enzyme was found as a homodimer, with the first 34 amino acids forming an efficient oligomerization domain that worked even in a different protein context. Using acetyl-CoA as donor substrate, OatC transferred acetyl groups exclusively onto polysialic acid joined by alpha2,9-linkages and did not act on free or CMP-activated sialic acid. Motif scanning revealed a nucleophile elbow motif (GXS286XGG), which is a hallmark of alpha/beta-hydrolase fold enzymes. In a comprehensive site-directed mutagenesis study, we identified a catalytic triad composed of Ser-286, Asp-376, and His-399. Consistent with a double-displacement mechanism common to alpha/beta-hydrolase fold enzymes, a covalent acetylenzyme intermediate was found. Together with secondary structure prediction highlighting an alpha/beta-hydrolase fold topology, our data provide strong evidence that OatC belongs to the alpha/beta-hydrolase fold family. This clearly distinguishes OatC from all other bacterial sialate O-acetyltransferases known so far because these are members of the hexapeptide repeat family, a class of acyltransferases that adopt a left-handed beta-helix fold and assemble into catalytic trimers.
Bergfeld
Anne K
AK
Department of Cellular Chemistry, Medical School Hannover, 30623 Hannover, Germany and the Institute for Hygiene and Microbiology, University of Wu¨rzburg, 97080 Wu¨rzburg, Germany.
Claus
Heike
H
Department of Cellular Chemistry, Medical School Hannover, 30623 Hannover, Germany and the Institute for Hygiene and Microbiology, University of Wu¨rzburg, 97080 Wu¨rzburg, Germany.
Lorenzen
Nina K
NK
Department of Cellular Chemistry, Medical School Hannover, 30623 Hannover, Germany and the Institute for Hygiene and Microbiology, University of Wu¨rzburg, 97080 Wu¨rzburg, Germany.
Spielmann
Fabian
F
Department of Cellular Chemistry, Medical School Hannover, 30623 Hannover, Germany and the Institute for Hygiene and Microbiology, University of Wu¨rzburg, 97080 Wu¨rzburg, Germany.
Vogel
Ulrich
U
Department of Cellular Chemistry, Medical School Hannover, 30623 Hannover, Germany and the Institute for Hygiene and Microbiology, University of Wu¨rzburg, 97080 Wu¨rzburg, Germany.
Mu Hlenhoff
Martina
M
Department of Cellular Chemistry, Medical School Hannover, 30623 Hannover, Germany and the Institute for Hygiene and Microbiology, University of Wu¨rzburg, 97080 Wu¨rzburg, Germany. Electronic address: muehlenhoff.martina@mh-hannover.de.
D000105:Acetyl Coenzyme A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000123:Acetyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs / Q000502:physiology; D016667:Bacterial Capsules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D006801:Humans; D019158:N-Acetylneuraminic Acid / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D038542:Neisseria meningitidis, Serogroup C / Q000201:enzymology* / Q000235:genetics; D017510:Protein Folding; D020836:Protein Structure, Quaternary / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; C018766:N-acylneuraminate-9(7)-O-acetyltransferase; D019158:N-Acetylneuraminic Acid