Overproduction of CMP-sialic acid synthetase for organic synthesis

Liu, Jennifer Lin Chun; Shen, Gwo‐Jenn; Ichikawa, Yoshitaka; Rutan, James F.; Zapata, Gerardo; Vann, Willie F.; Wong, Chi Huey

doi:10.1021/ja00036a044

Cited by 114 publications

(54 citation statements)

References 2 publications

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“…The final purification was done using a column (2 ϫ 25 cm) of Bio-Gel P-2 (Amersham Pharmacia Biotech) eluted with water following the usual procedure, and verified by polyacrylamide gel electrophoresis following the method of Troy and McCloskey (7). After hydrolysis by sulfuric acid (0.1 N H 2 SO 4 , 80°C), quantitation of sialic acid was determined by the thiobarbituric acid procedure as described previously (27).…”

Section: Methodsmentioning

confidence: 99%

Expression of α2,8/2,9-Polysialyltransferase fromEscherichia coli K92

Shen¹,

Datta²,

Izumi

et al. 1999

Journal of Biological Chemistry

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The capsular polysaccharide of Escherichia coli K92 contains alternating -8-NeuAc␣2-and -9-NeuAc␣2-linkages. The enzyme catalyzing this polymerizing reaction has been cloned from the genomic DNA of E. coli K92. The 1.2-kilobase polymerase chain reaction fragment was subcloned in pRSET vector and the protein was expressed in the BL21(DE3) strain of E. coli with a hexameric histidine at its N-terminal end. The enzyme was isolated in the supernatant after lysis of the cells and fractionated by ultracentrifugation. Western blotting using anti-histidine antibody showed the presence of a band that migrated at about 47.5 kDa on both reducing and nonreducing SDS-polyacrylamide gel electrophoresis, indicating a monomeric enzyme. Among the carbohydrate acceptors tested, N-acetylneuraminic acid and the gangliosides G D3 and G Q1b were preferred substrates. The cell-free enzyme reaction products obtained were characterized by NMR and mass spectrometry, which indicated the presence of both ␣2,9-and ␣2,8-linked polysialyl structure. The K92 neuS gene was used to transform the K1 strain of E. coli, the capsule of which contains only -8-NeuAc␣2-linkages. Analysis of the polysaccharides isolated from these transformed cells is consistent with the presence of both -8-NeuAc␣2-and -9-NeuAc␣2-linkages. Our results suggest that the neuS gene product of E. coli K92 catalyzes the synthesis of polysialic acid with ␣2,9-and ␣2,8-linkages in vitro and in vivo.

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Section: Methodsmentioning

confidence: 99%

Expression of α2,8/2,9-Polysialyltransferase fromEscherichia coli K92

Shen¹,

Datta²,

Izumi

et al. 1999

Journal of Biological Chemistry

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“…The panel of probes (Scheme 3) included terminus modifications such as the 9-azido-substituted derivative 4, [27] and two truncated Neu5Ac analogues with chain lengths of C 8 (2) and C 7 (3). [28] The importance of the amide moiety was tested using the 5-deaminated analogue KDN (5) [29] and its 5-epimer (6), [30] as well as by several variations of the N-acyl chain, such as substitution to glycolyl (7) [31] or azidoacetyl (8), [9] extension to 5-hexynoyl (9), introduction of arene moieties such as phenylacetyl and benzoyl (10/11), [32,33] or replace- [a] Data were determined by colorimetric pH-sensitive assay with varying concentrations of sialic acid substrates at a fixed CTP concentration of 0.5 mM, followed by Michaelis-Menten analysis.…”

mentioning

confidence: 99%

Flexibility of Substrate Binding of Cytosine‐5′‐Monophosphate‐N‐Acetylneuraminate Synthetase (CMP‐Sialate Synthetase) from Neisseria meningitidis: An Enabling Catalyst for the Synthesis of Neo‐sialoconjugates

Dong

Fessner

2011

Adv Synth Catal

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Abstract:We have successfully established a simple continuous colorimetric assay for the sensitive and reliable quantification of cytosine-5'-monophosphate-acetylneuraminate synthetase (CMP-sialate synthetase, CSS) activity based on the pH change of a released proton equivalent upon nucleotide activation of Neu5Ac or related analogues. Using this method, steady-state kinetic data of Neisseria meningitidis CSS were determined for cytosine-5'-triphosphate (CTP), N-acetylneuraminic acid (Neu5Ac) and eleven structural variations of the sialic acid, including backbone truncation, deamination, epimerization, and several N-acyl modifications. These data further demonstrate the unusual versatility of the N. meningitidis CSS for a general access to sialoconjugates containing non-natural sialic acid analogues. Remarkably, the assay allows covering a broad range of substrate parameters that span over more than three orders of magnitude for K M and k cat measurements. With the aid of a structural model built from X-ray crystal structure data, the kinetic data could be used to interpret potential protein contributions in substrate binding of Neu5Ac and its analogues. The Neu5Ac analogues were used for the preparation of neo-sialoconjugate analogues of 2,6-sialyllactose in a one-pot two-enzyme cascade, using N. meningitidis CSS together with the novel a2,6-sialyltransferase (a2,6SiaT) from Photobacterium leiognathi, which proved to be highly effective because of its optimum activity at alkaline pH, appropriately matching the requirements from the overall reaction system.

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“…5 Enzymatic modification of the sugar moiety of a glycoconjugate is another useful route to biologically important glycoconjugates. For example, the melanoma antigen 9-O-acetylganglioside GD3 can be prepared from GD3 via subtilisincatalyzed acetylation in dimethylformamide using vinyl acetate (81), or may be prepared from ganglioside GM3 via GD3 synthase (82)-catalyzed transfer of 9-O-acetylneuraminic acid from its CMP-derivative, which is in turn prepared enzymatically (83) as shown in Fig. 6.…”

Section: Chemoenzymatic Approaches To the Synthesis Of Complex Carbohmentioning

confidence: 99%

Intervention of carbohydrate recognition by proteins and nucleic acids.

Sears

Wong

1996

Proc. Natl. Acad. Sci. U.S.A.

113

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Carbohydrates in biological systems are often associated with specific recognition and signaling processes leading to important biological functions and diseases. Considerable efforts have been directed toward understanding and mimicking the recognition processes and developing effective agents to control the processes. The pace of discovery research in glycobiology and development of carbohydratebased therapeutics, however, has been relatively slow due to the lack of appropriate strategies and methods available for carbohydrate-related research. This review summarizes some of the most recent developments in the field, with particular emphasis on work from our laboratories regarding the use of chemoenzymatic strategies to tackle the carbohydrate recognition problem. Highlights include the study of selectincarbohydrate and aminoglycoside-RNA interactions and development of agents for the intervention of these recognition processes.Of the three major classes of biomolecules, carbohydrates perhaps are the least exploited. It has been known that carbohydrates can serve as structural components of natural products, as energy sources, or, more interestingly, as key elements in various molecular recognition processes, including bacterial and viral infections, cell adhesion in inflammation and metastasis, differentiation, development, regulation, and many other intercellular communication and signal transduction events (1). [Representative structures are presented in Fig. 1 (2-16).] The precise mechanism of many carbohydratemediated recognition processes are, however, not well understood, and, though potential opportunities exist, the pace of development of carbohydrate-based pharmaceuticals has been slower than that of the other classes of biomolecules. Several reasons may be attributed to this slow pace of development. First, some technical problems in the field are impossible or difficult to solve. There is no PCR equivalent replication system available for the amplification of minute amounts of carbohydrates to facilitate structure analysis and synthesis, and there is no machine available for the solid-phase synthesis of oligosaccharides to facilitate the study of their functions. In addition, synthesis of oligosaccharides in large quantities for therapeutic evaluation and development is in general very difficult and expensive. Second, carbohydrates generally possess undesirable properties for drug development. The affinity of carbohydrates for their protein receptors is relatively weak (17-21), with dissociation constants in the millimolar range, and carbohydrates are generally orally inactive and sensitive to enzymes in vivo. As a result, carbohydrates may only be used as injectable form for the treatment of acute symptoms.Understanding carbohydrate recognition is important, however, as this recognition process often occurs at the early stage of disease development, and intervention of such process may be beneficial. Furthermore, understanding the mechanism of carbohydrate recognition may lead to the devel...

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Overproduction of CMP-sialic acid synthetase for organic synthesis

Cited by 114 publications

References 2 publications

Expression of α2,8/2,9-Polysialyltransferase fromEscherichia coli K92

Expression of α2,8/2,9-Polysialyltransferase fromEscherichia coli K92

Flexibility of Substrate Binding of Cytosine‐5′‐Monophosphate‐N‐Acetylneuraminate Synthetase (CMP‐Sialate Synthetase) from Neisseria meningitidis: An Enabling Catalyst for the Synthesis of Neo‐sialoconjugates

Intervention of carbohydrate recognition by proteins and nucleic acids.

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