Engineering of a Cytidine 5′‐Monophosphate‐Sialic Acid Synthetase for Improved Tolerance to Functional Sialic Acids

Abstract

Sialic acid-containing glycoconjugates at the cell surface are of high importance in carbohydrate-mediated recognition phenomena in physiological and pathological events, as well as in bacterial or viral infection. A key step in the enzymatic synthesis of natural sialoconjugates and functional synthetic analogues is the activation of sialic acids to cytidine 5'-monophosphate (CMP)-sialic acid intermediates catalyzed by CMP-sialic acid synthetase (CSS). Based on our recently developed aligned protein model of substrate binding and a simple colorimetric screening assay, we have engineered the CSS from Neisseria meningitidis by structure-guided site-specific saturation mutagenesis at positions 192/193 to generate enzymes with broadened substrate scope. Top hits, including the F192S/F193Y variant, display an improvement of up to 70-fold catalytic efficiency relative to wild-type CSS for the conversion of sterically demanding N-acyl modified sialic acid analogues, without compromising protein stability. Such significantly enhanced substrate capacity is a major step forward to realizing a generalized chemo-enzymatic strategy for the efficient preparation of neo-sialoconjugate libraries, demonstrated by the highly efficient, regio-and stereospecific synthesis of 2,6-sialyllactose analogues by enzymatic coupling to the highly substrate tolerant a2,6-sialyltransferase from Photobacterium leiognathi JT-SHIZ-145. Our results further document the unusual versatility of the N. meningitidis CSS and engineered variants for a common synthetic approach to sialoconjugates comprising a large diversity of natural and non-natural sialic acid forms without the need for post-synthetic enzymatic modification.

An α2,3‐Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution

“…However,w en oted that isolated product yields were somewhat lower than anticipatedw hen compared to the complementaryr eactions using the P. leiognathi 2,6SiaT, [19,20] particularly when using only stoichiometric quantitieso fCTP.F or example, upon transfer of the parent Neu5Acalimiting maximum yield of 84 %r esulted for the labelled GM3, whereas the corresponding a2,6-sialylated trisaccharide had been obtained in 93 %y ield. [20] Likewise, the synthesis of unlabeled GM3 trisaccharide from lactose by 2,3SiaT pph catalysis proceeded with an incomplete yield of 86 %o nly.T his indicated that the 2,3-sialyltransfer was sufferingf rom ah ydrolytic uncoupling that was partially causing an unproductive consumptiono ft he limiting CTP quantity.I ndeed, many other 2,3SiaT and 2,6SiaTenzymes from the GT80 familyh ave been shown to display multifunctionality:B esides the desired sialyltransfer activity (A), promiscuous activitiess uch as CMP-Neu5Ac hydrolase (B;s ialyltransfer to water or phosphodiester hydrolysis) and sialidase (C,h ydrolytic cleavage of sialoside linkages) have been recorded (Scheme 2). [21,[24][25][26][27][28][29] Reversibility of sialyltransfer (A)w as recognized as the cause of ap urported trans-sialidase activity,w hich arises from transientC MP-Neu5Ac formation.…”

“…For synthetic studies, the purified 2,3SiaT pph was studied for its transfer activity with at est set of three natural (N-acetylneuraminic acid, 1, N-glycoloyl-neuraminic acid, 2,a nd KDN, 3) and three new-to-nature sialic acid derivatives (4-6,S cheme 1) that were availablef rom previouss tudies. [19,20] Sialic acids were activatedi ns itu by incubation with CSS from Neisseria meningitidis [23] in the presence of cytidinet riphosphate( CTP). Transfer to af luorophore-labeled lactoside 7 [19] was then monitored by HPLC, which showed the smooth conversion of all sialic acid compounds with generation of the expected labelled GM3 (Neu5Aca2-3Galb1-4Glc)t risaccharide 8 and the correspondinga nalogues 9-13 (Scheme 1).…”

“…[18] Previous work from our group has demonstratedt hat the 2,6SiaT from Photobacterium leiognathi JT-SHIZ-145 is aw idely useful catalystf or the preparative transfero fn ative and synthetic sialic acid derivatives to furnish the corresponding a2,6-linked sialoconjugates in high yield. [19,20] In continuation of these studies, we were interested in as uitable bacterial 2,3SiaTcatalyst for an effective approacht oward the regioisomeric a2,3linkeds ialoconjugates.…”

Recent Developments in Enzymatic Synthesis of Modified Sialic Acid Derivatives

“…A necessary step in such syntheses is to convert the sialic acid to its CMP derivative. Fortunately, N. meningitidis CMP‐sialic acid synthetase133 has broad substrate specificity toward the 5‐position of sialic acids28–30 compared to the same enzyme from other species (Figure 9). 134 Fessner and co‐workers reported a simple colorimetric assay for the quantification of CMP‐sialic acid synthetase activity based on the pH change due to the proton released upon nucleotide activation of sialic acids.…”

3.8.1 Designed Enzymatic Cascades

“…[84] Recently, the CMP sialic acid synthetase from Neisseria meningitidis has been engineered by site-specific saturation mutagenesis at positions 192/193 to generate mutants with improved catalytic efficiency relative to wild-type CMP sialic acid synthetase for the conversion of sterically demanding N-acyl-modified sialic acid analogues. [85] 3.8. 1.4.4 Tandem Reactions Employing Threonine-Dependent Aldolases Glycine-dependent aldolases catalyze the reversible aldol reaction of glycine with an aldehyde acceptor.…”