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

Abstract

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.

Direct Enzymatic Branch‐End Extension of Glycocluster‐Presented Glycans: An Effective Strategy for Programming Glycan Bioactivity

“…[12,13] Ester-protected lactosyla zide 22 was also synthesized as previously reported, [14] and the click reaction [15] with terminal acetylenes of scaffolds 8,12,15,17,19,a nd 21 was carriedo ut by using CuSO 4 ands odium ascorbate in DMF/H 2 O by MW at 80 8Cf or 15 min, to afford 23,25,27,29,31,a nd 33, with hydroxyl groups still protected. [12,13] Ester-protected lactosyla zide 22 was also synthesized as previously reported, [14] and the click reaction [15] with terminal acetylenes of scaffolds 8,12,15,17,19,a nd 21 was carriedo ut by using CuSO 4 ands odium ascorbate in DMF/H 2 O by MW at 80 8Cf or 15 min, to afford 23,25,27,29,31,a nd 33, with hydroxyl groups still protected.…”

“…[17,18] The pyrophosphate side product was hydrolyzed to inorganic phosphate in situ by adding ap yrophosphatase to prevent inhibition. [17,18] The pyrophosphate side product was hydrolyzed to inorganic phosphate in situ by adding ap yrophosphatase to prevent inhibition.…”

“…In the second stage, the two sets of glycoclustersw ere subsequently sialylated by using the a2,6-sialyltransferase of Photobacterium leiognathi (2,6SiaT) [17,19] as catalyst with either synthetic CMP-Neu5Ac or CMP-Neu5Gc as donor substrates, respectively.R eactions were carried out in Tris/HCl buffer (50 mm,p H8.6), supplemented with 50 mm MgCl 2 as cofactor, at 30 8Cw ith careful pH control. Five equivalents of the corresponding CMP-activated sialic acid were added as donor for each glycocluster valency to ensure complete conversion of each reactive site.…”

“…Five equivalents of the corresponding CMP-activated sialic acid were added as donor for each glycocluster valency to ensure complete conversion of each reactive site. [17] The progress of the reaction was monitored by HPLC.U pon complete conversion, reaction mixtures were centrifuged andl yophilized, andt he products were purified by gel permeation chromatography. [17] The progress of the reaction was monitored by HPLC.U pon complete conversion, reaction mixtures were centrifuged andl yophilized, andt he products were purified by gel permeation chromatography.…”

“…Minimum conformation structures of the glycoclusters were superimposed on the lactose bindings ite followed by as hort molecular dynamics run and energy minimization (see Experimental Section). By using this diversification tool, the conjugation of engineered sialic acid derivatives [8,17] makes astraightforward extension possible, which can be used to determine its effect on lectin binding affinitiesw ith our test set of glycoclusterso ro ther suitable scaffolds, [2] for VAAo ro ther biomedically relevant lectins. As showni nF igure 6, Ser200 and Gln238 form hydrogen bonds with 3-OH andt he carboxylic acid of the sialica cidr esidue, respectively.C learly,t hese interactions arrest the flexibility of the a2,6-sialylgalactosyl unit, counterbalancing their favorable contribution to the thermodynamic balance sheet.…”