Carbon-13 NMR investigation of the anomeric specificity of CMP-N-acetylneuraminic acid synthetase from Escherichia coli

Biochemistry ◽  
1992 ◽  
Vol 31 (3) ◽  
pp. 775-780 ◽  
Author(s):  
Michael G. Ambrose ◽  
Stephen J. Freese ◽  
Mary S. Reinhold ◽  
Thomas G. Warner ◽  
Willie F. Vann
Keyword(s):  
Escherichia Coli ◽  
Acetylneuraminic Acid ◽  
Anomeric Specificity

scholarly journals Sequence of the Cloned Escherichia coli K1 CMP-N-acetylneuraminic Acid Synthetase Gene

1989 ◽  
Vol 264 (25) ◽  
pp. 14769-14774
Author(s):  
G Zapata ◽  
W F Vann ◽  
W Aaronson ◽  
M S Lewis ◽  
M Moos
Keyword(s):  
Escherichia Coli ◽  
Acetylneuraminic Acid ◽  
Escherichia Coli K1

scholarly journals Novel serine/threonine-O-glycosylation with N-acetylneuraminic acid and 3-deoxy-D-manno-octulosonic acid by bacterial flagellin glycosyltransferases

Glycobiology ◽  
2020 ◽  
Author(s):  
Aasawari Khairnar ◽  
Sonali Sunsunwal ◽  
Ponnusamy Babu ◽  
T N C Ramya
Keyword(s):  
Escherichia Coli ◽  
Clostridium Botulinum ◽  
Associated Factors ◽  
Nucleotide Sugar ◽  
Acetylneuraminic Acid ◽  
Geobacillus Kaustophilus ◽  
Nonulosonic Acid ◽  
Glycosyl Donor ◽  
Pseudaminic Acid ◽  
Substrate Promiscuity

Abstract Some bacterial flagellins are O-glycosylated on surface-exposed serine/threonine residues with nonulosonic acids such as pseudaminic acid, legionaminic acid and their derivatives by flagellin nonulosonic acid glycosyltransferases, also called motility-associated factors (Maf). We report here two new glycosidic linkages previously unknown in any organism, serine/threonine-O-linked N-acetylneuraminic acid (Ser/Thr-O-Neu5Ac) and serine/threonine-O-linked 3-deoxy-D-manno-octulosonic acid or keto-deoxyoctulosonate (Ser/Thr-O-KDO), both catalyzed by Geobacillus kaustophilus Maf and Clostridium botulinum Maf. We identified these novel glycosidic linkages in recombinant G. kaustophilus and C. botulinum flagellins that were coexpressed with their cognate recombinant Maf protein in Escherichia coli strains producing the appropriate nucleotide sugar glycosyl donor. Our finding that both G. kaustophilus Maf (putative flagellin sialyltransferase) and C. botulinum Maf (putative flagellin legionaminic acid transferase) catalyzed Neu5Ac and KDO transfer on to flagellin indicates that Maf glycosyltransferases display donor substrate promiscuity. Maf glycosyltransferases have the potential to radically expand the scope of neoglycopeptide synthesis and posttranslational protein engineering.

scholarly journals Convergent Pathways for Utilization of the Amino Sugars N-Acetylglucosamine,N-Acetylmannosamine, and N-Acetylneuraminic Acid by Escherichia coli

1999 ◽  
Vol 181 (1) ◽  
pp. 47-54 ◽  
Author(s):  
Jacqueline Plumbridge ◽  
Eric Vimr
Keyword(s):  
Escherichia Coli ◽  
Carbon Sources ◽  
Amino Sugars ◽  
Good Growth ◽  
Gene Products ◽  
Phosphotransferase System ◽  
Regulatory Mutation ◽  
Putative Gene ◽  
Acetylneuraminic Acid ◽  
K 12

ABSTRACT N-Acetylglucosamine (GlcNAc) andN-acetylneuraminic acid (NANA) are good carbon sources forEscherichia coli K-12, whereasN-acetylmannosamine (ManNAc) is metabolized very slowly. The isolation of regulatory mutations which enhanced utilization of ManNAc allowed us to elucidate the pathway of its degradation. ManNAc is transported by the manXYZ-encoded phosphoenolpyruvate-dependent phosphotransferase system (PTS) transporter producing intracellular ManNAc-6-P. This phosphorylated hexosamine is subsequently converted to GlcNAc-6-P, which is further metabolized by the nagBA-encoded deacetylase and deaminase of the GlcNAc-6-P degradation pathway. Two independent mutations are necessary for good growth on ManNAc. One mutation maps tomlc, and mutations in this gene are known to enhance the expression of manXYZ. The second regulatory mutation was mapped to the nanAT operon, which encodes the NANA transporter and NANA lyase. The combined action of thenanAT gene products converts extracellular NANA to intracellular ManNAc. The second regulatory mutation defines an open reading frame (ORF), called yhcK, as the gene for the repressor of the nan operon (nanR). Mutations in the repressor enhance expression of the nanAT genes and, presumably, three distal, previously unidentified genes,yhcJIH. Expression of just one of these downstream ORFs,yhcJ, is necessary for growth on ManNAc in the presence of an mlc mutation. The yhcJ gene appears to encode a ManNAc-6-P-to-GlcNAc-6-P epimerase (nanE). Another putative gene in the nan operon, yhcI, likely encodes ManNAc kinase (nanK), which should phosphorylate the ManNAc liberated from NANA by the NanA protein. Use of NANA as carbon source by E. coli also requires thenagBA gene products. The existence of a ManNAc kinase and epimerase within the nan operon allows us to propose that the pathways for dissimilation of the three amino sugars GlcNAc, ManNAc, and NANA, all converge at the step of GlcNAc-6-P.

The anomeric specificity of β-galactosidase and lac permease from Escherichia coli

1981 ◽  
Vol 59 (2) ◽  
pp. 100-105 ◽  
Author(s):  
R. E. Huber ◽  
K. L. Hurlburt ◽  
C. L. Turner
Keyword(s):  
Escherichia Coli ◽  
Glycosidic Bond ◽  
Lac Operon ◽  
Acceptor Reaction ◽  
Glucose Release ◽  
Bond Breakage ◽  
Lactose Metabolism ◽  
Anomeric Specificity

β-Galactosidase was found to act on α-lactose slightly more than twice as rapidly as on β-lactose for both the hydrolysis and transgalactosylis reactions. The effect was shown to be on the Vmax values; the Km values for the different anomeric forms were the same. The step of the reaction for which the enzyme has anomeric specificity was shown to be glycosidic bond breakage. The steps in glucose release or in the glucose acceptor reaction were not affected by anomeric composition. Neither allolactose hydrolysis nor transport of lactose into the cells by lac permease was sensitive to the anomeric composition of the substrate. The implications of these results for lac operon induction and for lactose metabolism are discussed.

Structure of the O56 antigen of Escherichia coli, a polysaccharide containing 7-substituted α-N-acetylneuraminic acid

1993 ◽  
Vol 238 ◽  
pp. 261-270 ◽  
Author(s):  
Grigorij Kogan ◽  
Alexander S. Shashkov ◽  
Barbara Jann ◽  
Klaus Jann
Keyword(s):  
Escherichia Coli ◽  
Acetylneuraminic Acid

scholarly journals The role of cysteine residues 129 and 329 in Escherichia coli K1 CMP-NeuAc synthase

1993 ◽  
Vol 295 (2) ◽  
pp. 485-491 ◽  
Author(s):  
G Zapata ◽  
P P Roller ◽  
J Crowley ◽  
W F Vann
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Acetylneuraminic Acid ◽  
Denatured States ◽  
Escherichia Coli K1

N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.

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