Abstract
The acquisition of host-derived sialic acid is an important virulence factor for some bacterial pathogens, but in vivo this sugar acid is sequestered in sialoconjugates as the alpha-anomer. In solution, however, sialic acid is present mainly as the beta-anomer, formed by a slow spontaneous mutarotation. We studied the Escherichia coli protein YjhT as a member of a family of uncharacterized proteins present in many sialic acid-utilizing pathogens. This protein is able to accelerate the equilibration of the alpha- and beta-anomers of the sialic acid N-acetylneuraminic acid, thus describing a novel sialic acid mutarotase activity. The structure of this periplasmic protein, solved to 1.5A resolution, reveals a dimeric 6-bladed unclosed beta-propeller, the first of a bacterial Kelch domain protein. Mutagenesis of conserved residues in YjhT demonstrated an important role for Glu-209 and Arg-215 in mutarotase activity. We also present data suggesting that the ability to utilize alpha-N-acetylneuraminic acid released from complex sialoconjugates in vivo provides a physiological advantage to bacteria containing YjhT.
Highlights
FEBRUARY 22, 2008 VOLUME 283 NUMBER 8 the intestinal bacterium Escherichia coli can grow on Neu5Ac as a sole carbon source [4]
The intestinal mucous layer is rich in sialic acids, and E. coli genes required for sialic acid catabolism are induced in this environment and are important for colonization [5]
H. influenzae is unable to synthesize Neu5Ac de novo and must acquire it from the host [9, 11]. This has recently been demonstrated to be dependent on a tripartite ATP-independent periplasmic (TRAP) transporter [12,13,14], which is encoded in a genomic region that contains the genes for sialic acid catabolism [1, 12]
Summary
Cloning and Expression of Native and Mutant YjhT-His Proteins—The yjhT gene was amplified from genomic DNA of E. coli K12 MG1655 using KOD Hot Start polymerase (EMD Biosciences) and primers EcyjhT-NdeI and EcyjhT-XhoI (supplemental Table 1). Crystallization Procedure—Purified SeMet-substituted YjhT was dialyzed against double distilled water and concentrated to 12 mg/ml. Data Collection and Structure Solution—A single wavelength data set extending to a resolution of 1.5 Å with a high redundancy was collected from a crystal of SeMet substituted YjhT at the ESRF, Grenoble, France, on beamline ID14-4 (Table 1). Refinement of the selenium atom positions using the program MLPHARE provided experimental phase probability distributions in terms of the Hendrikson-Lattman coefficients. These served as restraints during the ARP/wARP-REFMAC model building process [22], which led to a model with 587 of 714 residues built and sequence coverage of 99%. Reflections (working/free) Outer shell R-factorc (R-freed) Outer shell reflections (working/free)e
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