Regeneration of PAPS for the Enzymatic Synthesis of Sulfated Oligosaccharides

Burkart, Michael D.; Izumi, Masayuki; Chapman, Eli; Lin, Chun Hung; Wong, Chi Huey

doi:10.1021/jo000266o

Cited by 98 publications

(91 citation statements)

References 48 publications

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“…Chitobiose was arbitrarily chosen as the integer value because of its low specificity constant with respect to the other three chitooligomers. The relative reaction specificity of the four substrates determined by our assay was in excellent agreement with that reported in the literature [31,37], which suggests that in the pathway of Nod signal biosynthesis, the lipochitotetrasaccharide Nod factor is preferably sulfated by NodH.…”

Section: Individual Kinetic Measurementssupporting

confidence: 89%

“…Employing a thin layer chromatography assay, Long et al [30] and Kondorosi and coworkers [31] discovered that NodST can sulfate biosynthetic chitooligosaccharides in addition to natural Nod factors, although with a lower reaction activity. Kinetic analyses with both Nod factors and chitooligosaccharides of different chain lengths revealed the highest binding affinity and best reaction activity of NodH for the appropriate tetrameric substrates, which guarantees the modification of the tetrameric Nod factor-the most active one in Rhizobium meliloti, and thus contributes to host specificity [31,37]. Kinetic constants for various chitooligosaccharides have been determined individually by Kondorosi and coworkers [31] and Wong et al [37], which indicated that chitotetraose is a better substrate than chitotriose, chitopentaose, or chitohexaose, while NodST's activity for chitobiose is more than one order of magnitude lower than that for the other four.…”

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confidence: 95%

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Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

Leary

2004

J. Am. Soc. Mass Spectrom.

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The traditional method used to investigate the reaction specificity of an enzyme with different substrates is to perform individual kinetic measurements. In this case, a series of varied concentrations are required to study each substrate and a non-regression analysis program is used several times to obtain all the specificity constants for comparison. To avoid the large amount of experimental materials, long analysis time, and redundant data processing procedures involved in the traditional method, we have developed a novel strategy for rapid determination of enzyme substrate specificity using one reaction system containing multiple competing substrates. In this multiplex assay method, the electrospray ionization mass spectrometry (ESI-MS) technique was used for simultaneous quantification of multiple products and a steady-state kinetics model was established for efficient specificity constant calculation. The system investigated was the bacterial sulfotransferase NodH (NodST), which is a host specific nod gene product that catalyzes the sulfate group transfer from 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS) to natural Nod factors or synthetic chitooligosaccharides. Herein, the reaction specificity of NodST for four chitooligosaccharide acceptor substrates of different chain length (chitobiose, chitotriose, chitotetraose, and chitopentaose) was determined by both individual kinetic measurements and the new multiplex ESI-MS assay. The results obtained from the two methods were compared and found to be consistent. The multiplex ESI-MS assay is an accurate and valid method for substrate specificity evaluation, in which multiple substrates can be evaluated in one assay. (J Am Soc Mass Spectrom 2004, 15, 233-243)

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Section: Individual Kinetic Measurementssupporting

confidence: 89%

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confidence: 95%

Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

Leary

2004

J. Am. Soc. Mass Spectrom.

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“…First, highly active HS O-sulfotransferases were expressed in E. coli, in order to provide sufficient quantities of sulfotransferases. Second, the sulfotransfer reactions were coupled with the PAPS regeneration system that was developed by the Wong group (56). This strategy eliminates the inhibition effect of PAP.…”

Section: Chemoenzymatic Synthesis Of Hs With Biological Activitiesmentioning

confidence: 99%

“…The PAPS regeneration system utilizes arylsulfotransferase IV, which can converts PAP to PAPS as illustrated in Figure 6B. In addition, the PAPS regeneration system utilizes the p-nitrophenol sulfate PNPS as the sulfo donor and requires only catalytic amounts of PAP, thereby reducing the cost of synthesis by more than 1000-fold (56). Immobilized enzymes are used in this approach for reuse and improved thermal stability of the HS sulfotransferases.…”

Section: Chemoenzymatic Synthesis Of Hs With Biological Activitiesmentioning

confidence: 99%

Anticoagulant heparan sulfate: structural specificity and biosynthesis

Liu

Pedersen

2007

Appl Microbiol Biotechnol

123

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SummaryHeparan sulfate (HS) is present on the surface of endothelial and surrounding tissues in large quantities. It plays important roles in regulating numerous functions of the blood vessel wall, including blood coagulation, inflammation response and cell differentiation. HS is a highly sulfated polysaccharide containing glucosamine and glucuronic/iduronic acid repeating disaccharide units. The unique sulfated saccharide sequences of HS determine its specific functions. Heparin, an analogue of heparan sulfate, is the most commonly used anticoagulant drug. Because of its wide range of biological functions, HS has become an interesting molecule to biochemists, medicinal chemists and developmental biologists. Here, we summarize recent progress towards understanding the interaction between heparan sulfate and blood coagulating factors, the biosynthesis of anticoagulant heparan sulfate and the mechanism of action of heparan sulfate biosynthetic enzymes. Further, knowledge of the biosynthesis of HS facilitates the development of novel enzymatic approaches to synthesize HS from bacterial capsular polysaccharides and to produce polysaccharide end products with high specificity for the biological target. These advancements provide the foundation for the development of polysaccharide-based therapeutic agents.

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“…Next, modification reactions are undertaken relying on chemical deprotection and modification, and enzymatic modification using sulfotransferases and epimerase. Since half of the mass of heparin is sodium sulfate and the remainder is carbohydrate, a large amount of sulfo donor 3 0 -phosphoadenosine-5 0 -phosphosulfate (PAPS) is required, and as reactions are scaled up, this typically requires cofactor recycling (Burkart et al 2000; Fig. 1).…”

Section: Synthetic Methods Strategymentioning

confidence: 99%