Dataset Information

Structural and enzymatic characterization of NanS (YjhS), a 9-O-Acetyl N-acetylneuraminic acid esterase from Escherichia coli O157:H7.

ABSTRACT: There is a high prevalence of sialic acid in a number of different organisms, resulting in there being a myriad of different enzymes that can exploit it as a fermentable carbon source. One such enzyme is NanS, a carbohydrate esterase that we show here deacetylates the 9 position of 9-O-sialic acid so that it can be readily transported into the cell for catabolism. Through structural studies, we show that NanS adopts a SGNH hydrolase fold. Although the backbone of the structure is similar to previously characterized family members, sequence comparisons indicate that this family can be further subdivided into two subfamilies with somewhat different fingerprints. NanS is the founding member of group II. Its catalytic center contains Ser19 and His301 but no Asp/Glu is present to form the classical catalytic triad. The contribution of Ser19 and His301 to catalysis was confirmed by mutagenesis. In addition to structural characterization, we have mapped the specificity of NanS using a battery of substrates.

SUBMITTER: Rangarajan ES

PROVIDER: S-EPMC3149194 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Structural and enzymatic characterization of NanS (YjhS), a 9-O-Acetyl N-acetylneuraminic acid esterase from Escherichia coli O157:H7.

Rangarajan Erumbi S ES Ruane Karen M KM Proteau Ariane A Schrag Joseph D JD Valladares Ricardo R Gonzalez Claudio F CF Gilbert Michel M Yakunin Alexander F AF Cygler Miroslaw M

Protein science : a publication of the Protein Society 20110531 7

There is a high prevalence of sialic acid in a number of different organisms, resulting in there being a myriad of different enzymes that can exploit it as a fermentable carbon source. One such enzyme is NanS, a carbohydrate esterase that we show here deacetylates the 9 position of 9-O-sialic acid so that it can be readily transported into the cell for catabolism. Through structural studies, we show that NanS adopts a SGNH hydrolase fold. Although the backbone of the structure is similar to prev ...[more]

PMID: 21557376

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Project description:The sialic acids (N-acylneuraminates) are a group of nine-carbon keto-sugars existing mainly as terminal residues on animal glycoprotein and glycolipid carbohydrate chains. Bacterial commensals and pathogens exploit host sialic acids for nutrition, adhesion, or antirecognition, where N-acetyl- or N-glycolylneuraminic acids are the two predominant chemical forms of sialic acids. Each form may be modified by acetyl esters at carbon position 4, 7, 8, or 9 and by a variety of less-common modifications. Modified sialic acids produce challenges for colonizing bacteria, because the chemical alterations to N-acetylneuraminic acid (Neu5Ac) confer increased resistance to sialidase and aldolase activities essential for the catabolism of host sialic acids. Bacteria with O-acetyl sialate esterase(s) utilize acetylated sialic acids for growth, thereby gaining a presumed metabolic advantage over competitors lacking this activity. Here, we demonstrate the esterase activity of Escherichia coli NanS after purifying it as a C-terminal HaloTag fusion. Using a similar approach, we show that E. coli strain O157:H7 Stx prophage or prophage remnants invariably include paralogs of nanS often located downstream of the Shiga-like toxin genes. These paralogs may include sequences encoding N- or C-terminal domains of unknown function where the NanS domains can act as sialate O-acetyl esterases, as shown by complementation of an E. coli strain K-12 nanS mutant and the unimpaired growth of an E. coli O157 nanS mutant on O-acetylated sialic acid. We further demonstrate that nanS homologs in Streptococcus spp. also encode active esterase, demonstrating an unexpected diversity of bacterial sialate O-acetyl esterase.The sialic acids are a family of over 40 naturally occurring 9-carbon keto-sugars that function in a variety of host-bacterium interactions. These sugars occur primarily as terminal carbohydrate residues on host glycoproteins and glycolipids. Available evidence indicates that diverse bacterial species use host sialic acids for adhesion or as sources of carbon and nitrogen. Our results show that the catabolism of the diacetylated form of host sialic acid requires a specialized esterase, NanS. Our results further show that nanS homologs exist in bacteria other than Escherichia coli, as well as part of toxigenic E. coli prophage. The unexpected diversity of these enzymes suggests new avenues for investigating host-bacterium interactions. Therefore, these original results extend our previous studies of nanS to include mucosal pathogens, prophage, and prophage remnants. This expansion of the nanS superfamily suggests important, although as-yet-unknown, functions in host-microbe interactions.

Project description:Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL933 encodes the single chromosomal 9-O-acetylesterase NanS, and several copies of prophage-encoded 9-O-acetylesterases (NanS-p). These enzymes have recently been shown to cleave 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac2) to yield de-O-acetylated Neu5Ac, the latter of which may serve as a carbon and/or nitrogen source. In the current study, we investigated the NanS- and NanS-p-mediated digestion of synthetic O-acetylated neuraminic acids and bovine submaxillary glands mucin (BSM)-derived O-acetylneuraminic acids by high-performance thin-layer chromatography (HPTLC) and nano electrospray ionization mass spectrometry (nanoESI MS). Initial HPTLC analyses showed the expected activity of NanS and NanS-p variants for Neu5,9Ac2. However, all tested enzymes were unable to de-O-acetylate 5-N-acetyl-4-O-acetylneuraminic acid (Neu5,4 Ac2) in our test system. The nanoESI MS analysis of neuraminic acids after treatment of BSM with NanS-p gave evidence that NanS-p variants of EHEC O157:H7 strain EDL933 cleave off O-acetyl groups from mono-, di-, and tri-O-acetylated Neu5Ac and N-glycolylneuraminic acid (Neu5Gc), regardless of the carbon positions C7, C8 or C9 of the acetate esters. This enzyme activity leads to neuraminidase-accessible Neu5Ac and Neu5Gc on mucin glycans. Moreover, we could demonstrate by HPTLC analyses that recombinant Bacteroides thetaiotaomicron sialidase (BTSA-His) was able to cleave Neu5Ac and Neu5,9Ac2 from BSM and that the combination of BTSA-His with both NanS-His and NanS-p-His derivatives enhanced the release of de-O-acetylated core Neu5Ac and Neu5Gc from mammalian mucin O-glycans. Growth experiments with EHEC wildtype strain EDL933, its nanS and nanS/nanS-p1a-p7 mutant and exogenous BTSA-His in BSM demonstrated that the presence of BTSA-His enhanced growth of EDL933 and the nanS deletion mutant but not the nanS/nanS-p1a-p7 mutant. Thus, we hypothesize that the expression of sialic acid O-acetylesterases with a broad specificity could be an advantage in competition with the gut microbiota for nutrients and facilitate EHEC colonization in the human large intestine.

Dataset Information

Structural and enzymatic characterization of NanS (YjhS), a 9-O-Acetyl N-acetylneuraminic acid esterase from Escherichia coli O157:H7.

Publications

Structural and enzymatic characterization of NanS (YjhS), a 9-O-Acetyl N-acetylneuraminic acid esterase from Escherichia coli O157:H7.

Similar Datasets

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