Chemoenzymatic synthesis of CMP–sialic acid derivatives by a one-pot two-enzyme system: comparison of substrate flexibility of three microbial CMP–sialic acid synthetases

Yu, Hai; Yu, Hui; Karpel, Rebekah; Chen, Xi

doi:10.1016/j.bmc.2004.09.030

Cited by 219 publications

(315 citation statements)

References 47 publications

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“…Sialyl glycans containing various natural modifications of sialic acid linked ␣2-3 or ␣2-6 to the three different fluorescent glycans described above were synthesized using a highly efficient one-pot three-enzyme system similar to that reported previously (19,20,25,28). The fluorescent glycan-AEAB conjugates (GAEABs) of LNT, LNnT, or NA2 (130 -200 g) were incubated with three enzymes, including Escherichia coli sialic acid aldolase (28) (10 -15 g), Neisseria meningitidis CMPsialic acid synthetase (28) (10 -15 g), and Photobacterium damselae ␣2-6-sialyltransferase (25) (10 -15 g; used for the synthesis of ␣2-6-linked sialosides) or Pasteurella multocida ␣2-3-sialyltransferase PmST1 (19) (3-6 g; used for the synthesis of ␣2-3-linked sialosides) in a reaction mixture of 25 l containing 100 mM Tris-HCl buffer, pH 8.5.…”

Section: Methodsmentioning

confidence: 99%

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A Sialylated Glycan Microarray Reveals Novel Interactions of Modified Sialic Acids with Proteins and Viruses

Song

Chen

et al. 2011

Journal of Biological Chemistry

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Many glycan-binding proteins in animals and pathogens recognize sialic acid or its modified forms, but their molecular recognition is poorly understood. Here we describe studies on sialic acid recognition using a novel sialylated glycan microarray containing modified sialic acids presented on different glycan backbones. Glycans terminating in ␤-linked galactose at the nonreducing end and with an alkylamine-containing fluorophore at the reducing end were sialylated by a one-pot three-enzyme system to generate ␣2-3-and ␣2-6-linked sialyl glycans with 16 modified sialic acids. The resulting 77 sialyl glycans were purified and quantified, characterized by mass spectrometry, covalently printed on activated slides, and interrogated with a number of key sialic acid-binding proteins and viruses. Sialic acid recognition by the sialic acid-binding lectins Sambucus nigra agglutinin and Maackia amurensis lectin-I, which are routinely used for detecting ␣2-6-and ␣2-3-linked sialic acids, are affected by sialic acid modifications, and both lectins bind glycans terminating with 2-keto-3-deoxy-D-glycero-D-galactonononic acid (Kdn) and Kdn derivatives stronger than the derivatives of more common N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Three human parainfluenza viruses bind to glycans terminating with Neu5Ac or Neu5Gc and some of their derivatives but not to Kdn and its derivatives. Influenza A virus also does not bind glycans terminating in Kdn or Kdn derivatives. An especially novel aspect of human influenza A virus binding is its ability to equivalently recognize glycans terminated with either ␣2-6-linked Neu5Ac9Lt or ␣2-6-linked Neu5Ac. Our results demonstrate the utility of this sialylated glycan microarray to investigate the biological importance of modified sialic acids in protein-glycan interactions.

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Section: Methodsmentioning

confidence: 99%

“…acetyl or lactyl group). The following were added to this mixture: (a) sialic acid precursors (20,25,28,29) (e.g. mannose, N-acetylmannosamine, or their derivatives) at 2.0 or 4.0 eq relative to LNT or LNnT and NA2 (biantennary-AEAB conjugate), respectively; (b) sodium pyruvate (5 eq for LNT or LNnT, 10 equiv.…”

Section: Methodsmentioning

confidence: 99%

A Sialylated Glycan Microarray Reveals Novel Interactions of Modified Sialic Acids with Proteins and Viruses

Song

Chen

et al. 2011

Journal of Biological Chemistry

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136

115

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“…It can also be used with sialidase to determine the total amount of sialic acid in glycoproteins, glycolipids, polysialic acids, and cell surfaces (Kolisis 1986). Synthetically, NanA has been broadly used in the aldol addition direction for the synthesis of Neu5Ac and its analogs (Huang et al 2007;Wong and Whitesides 1994;Yu et al 2004). …”

Section: Reactionmentioning

confidence: 99%

“…Q9CKB0) has been identified (Steenbergen et al 2005). Previously, we reported the cloning of a NanA from E. coli K-12 substrain MG1655 (EcNanA) (Yu et al 2004). The EcNanA has been used in one-pot multiple-enzyme systems to produce CMP-sialic acids, sialosides, and their analogs (Yu et al 2005;Yu et al 2004).…”

Section: Reactionmentioning

confidence: 99%

“…Previously, we reported the cloning of a NanA from E. coli K-12 substrain MG1655 (EcNanA) (Yu et al 2004). The EcNanA has been used in one-pot multiple-enzyme systems to produce CMP-sialic acids, sialosides, and their analogs (Yu et al 2005;Yu et al 2004). It has also been used in the synthesis of disaccharides containing a sialic acid at the reducing end (Huang et al 2007;.…”

Section: Reactionmentioning

confidence: 99%

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Pasteurella multocida sialic acid aldolase: a promising biocatalyst

Cao

et al. 2008

Appl Microbiol Biotechnol

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Sialic acid aldolases or N-acetylneuraminate lyases (NanAs) catalyze the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) to form pyruvate and N-acetyl-D-mannosamine (ManNAc). A capillary electrophoresis (CE) assay was developed to directly characterize the activities of NanAs in both Neu5Ac cleavage and Neu5Ac synthesis directions. The assay was used to obtain the pH profile and the kinetic data of a NanA cloned from Pasteurella multocida P-1059 (PmNanA) and a previously reported recombinant Escherichia coli K12 NanA (EcNanA). Both enzymes are active in a broad pH range of 6.0-9.0 in both reaction directions and have similar kinetic parameters. Substrates specificity studies showed that 5-O-methyl-ManNAc, a ManNAc derivative, can be used efficiently as a substrate by PmNanA, but not efficiently by EcNanA, for the synthesis of 8-O-methyl Neu5Ac. In addition, PmNanA (250 mg per liter culture) has a higher expression level (2.5 fold) than EcNanA (94 mg per liter culture). The higher expression level and a broader substrate tolerance make PmNanA a better catalyst than EcNanA for the chemoenzymatic synthesis of sialic acids and their derivatives.

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Combinatorial Biosynthesis of Complex Carbohydrates

Chen

2011

Carbohydrate Recognition

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Chemoenzymatic synthesis of CMP–sialic acid derivatives by a one-pot two-enzyme system: comparison of substrate flexibility of three microbial CMP–sialic acid synthetases

Cited by 219 publications

References 47 publications

A Sialylated Glycan Microarray Reveals Novel Interactions of Modified Sialic Acids with Proteins and Viruses

A Sialylated Glycan Microarray Reveals Novel Interactions of Modified Sialic Acids with Proteins and Viruses

Pasteurella multocida sialic acid aldolase: a promising biocatalyst

Combinatorial Biosynthesis of Complex Carbohydrates

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