Amino acid 310 determines the donor substrate specificity of serogroup W-135 and Y capsule polymerases of Neisseria meningitidis.
The capsular polysaccharides of serogroup W-135 and Y meningococci are sialic acid-containing heteropolymers, with either galactose or glucose as the second sugar residue. As shown previously, sequences of the predicted enzymes that catalyse capsule polymerization, i.e. SiaD(W-135) and SiaD(Y), differ in only a few amino acids. By in vitro assays with purified recombinant proteins, SiaD(W-135) and SiaD(Y) were now confirmed to be the capsule polymerases harbouring both hexosyltransferase and sialyltransferase activity. In order to identify amino acids crucial for substrate specificity of the capsule polymerases, polymorphic sites were narrowed down by DNA sequence comparisons and subsequent site-directed mutagenesis. Serogroup-specific amino acids were restricted to the N-terminal part of the proteins. Exclusively amino acid 310, located within the nucleotide recognition domain of the enzymes' predicted hexosyltransferase moiety, accounted for substrate specificity as shown by immunochemistry and in vitro activity assay. Pro-310 determined galactosyltransferase activity that resulted in a serogroup W-135 capsule and Gly-310 determined glucosyltransferase activity that resulted in a serogroup Y capsule. In silico analysis revealed a similar amino acid-based association in other members of the same glycosyltransferase family irrespective of the bacterial species.
Claus
Heike
H
Institut für Hygiene und Mikrobiologie, Universität Würzburg, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. hclaus@hygiene.uni-wuerzburg.de
Stummeyer
Katharina
K
Batzilla
Julia
J
Mühlenhoff
Martina
M
Vogel
Ulrich
U
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D016667:Bacterial Capsules / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D038582:Neisseria meningitidis, Serogroup W-135 / Q000201:enzymology* / Q000235:genetics; D038581:Neisseria meningitidis, Serogroup Y / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids; D011110:Polymorphism, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D000596:Amino Acids; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D006602:Hexosyltransferases