Enzyme Module Systems for the Synthesis of Uridine 5′‐Diphospho‐α‐D‐glucuronic Acid and Non‐Sulfated Human Natural Killer Cell‐1 (HNK‐1) Epitope

Abstract

Tailor-made strategies for the stereo-and regioselective multi-step enzymatic synthesis of glycoconjugates require well characterized glycosyltransferases andc arbohydrate modifying enzymes. We here report on an ovel enzyme cascade for the synthesiso fu ridine 5'-diphospho-a-d-glucuronic acid (UDP-GlcA) andt he non-sulfated human natural killer cell-1 (HNK-1) epitope including in situ regenerationo fU DP-GlcA andt he cofactor nicotinamide adenine dinucleotide NAD + + by the combination of four enzymes in one-pot.I nt he first enzyme module sucroses ynthase 1( SuSy1) is usedt op roduce uridine 5'-diphospho-a-d-glucose (UDP-Glc) from sucrose and uridine 5'-diphosphate (UDP). Thec ombination with UDP-Glc dehydrogenase in the second enzyme module leads to the synthesiso fU DP-GlcA with concomitant in situ regeneration of the cofactor NAD + + by nicotinamide adenine dinucleotide hydride (NADH)-oxidase.I nt he third enzyme module the mammalian glucuronyltransferase GlcAT-Pc atalyzes the synthesiso ft he non-sulfated HNK-1e pitope by regioselective transfer of GlcA onto N-acetyllactosamine type 2( LacNAc type 2). We present ac omprehensive studyo ns ubstrate kinetics,s ubstrate specificities,v ariation and relation of enzyme activitiesa s well as cross inhibition of intermediate products.With optimized reactionc onditions we obtain superior product yieldsw ith streamlined synthesisc osts for the expensiven ucleotide sugar UDP-GlcA and cofactor NAD + + .

In Vitro One‐Pot Enzymatic Synthesis of Hyaluronic Acid from Sucrose and N‐Acetylglucosamine: Optimization of the Enzyme Module System and Nucleotide Sugar Regeneration

“…[13] For HA synthesis, we replaced human UGDH by the novel recombinant szUGDH (HasB) owing to its highers pecific activity with 0.6 Umg À1 (TableS1i nt he SupportingI nformation) versus0.1 Umg À1 , [13] both in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-NaOH buffer, pH 7.5, and at 25 8C. [13] For HA synthesis, we replaced human UGDH by the novel recombinant szUGDH (HasB) owing to its highers pecific activity with 0.6 Umg À1 (TableS1i nt he SupportingI nformation) versus0.1 Umg À1 , [13] both in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-NaOH buffer, pH 7.5, and at 25 8C.…”

“…[13] For HA synthesis, we replaced human UGDH by the novel recombinant szUGDH (HasB) owing to its highers pecific activity with 0.6 Umg À1 (TableS1i nt he SupportingI nformation) versus0.1 Umg À1 , [13] both in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-NaOH buffer, pH 7.5, and at 25 8C. Consistent with data on UGDH from other organisms, [13,18] the optimal activity of szUGDH was found in the alkaliner ange between pH 8a nd 10, depending on the buffer system ( Figure S3 in the Supporting Information). Consistent with data on UGDH from other organisms, [13,18] the optimal activity of szUGDH was found in the alkaliner ange between pH 8a nd 10, depending on the buffer system ( Figure S3 in the Supporting Information).…”

Toward Automated Enzymatic Glycan Synthesis in a Compartmented Flow Microreactor System

“…The enzyme production was done as described in our previous studies by Engels et al [15] for UDP-glucose-dehydrogenase (UGDH, EC 1.1.1.22), NADH oxidase (NOX, EC 1.6.99.-) and β1,3glucuronyltransferase (β3GlcAT, EC 2.4.1.17, and by Wahl et al [13] for galactokinase (GalK, EC 2.7.1.6) and for UDP-sugar pyrophosphorylase (USP, EC 2.7.7.64), [12] and by Fischöder et al [14] for β1,4galactosyltransferase (β4GalT, EC 2.4.1.38). The enzyme production was done as described in our previous studies by Engels et al [15] for UDP-glucose-dehydrogenase (UGDH, EC 1.1.1.22), NADH oxidase (NOX, EC 1.6.99.-) and β1,3glucuronyltransferase (β3GlcAT, EC 2.4.1.17, and by Wahl et al [13] for galactokinase (GalK, EC 2.7.1.6) and for UDP-sugar pyrophosphorylase (USP, EC 2.7.7.64), [12] and by Fischöder et al [14] for β1,4galactosyltransferase (β4GalT, EC 2.4.1.38).…”

“…Details of the IR-unit for preheating and the TCM are discussed by Heinzler et al, [11] describing the reactor system. To test different temperatures, the activity of 15 mg/mL MEB was investigated at the respective optimal pH value of the free enzymes according to literature (UGDH/NOX/ GalK, [13] USP, [12] GalT, [14] GlcAT [15] ). For each assay, 15 μg of MEB with a loading of 15 mg immobilized enzyme per mL settled beads were used in a 100 μL compartment.…”

“…[12] In the EM-UDP-GlcA, UDP-glucose-dehydrogenase (UGDH) generates Uridine 5'-diphosphate-glucuronic acid (UDP-GlcA, 5) from UDP-glucose (UDP-Glc, 4), while NADH oxidase (NOX) regenerates the co-substrate NAD + . [15] In this work, we demonstrate automated enzymatic glycan synthesis by enzyme cascade reactions, conducted in the CFMS, using six different enzymes immobilized on magnetic particles (Scheme 1A). [14] The UDP-GlcA, produced in the EM-UDP-GlcA, was used for glucuronosyltransferase (GlcAT) to transfer the GlcA onto LacNAclinker-tBoc in the EM-GlcAT, resulting in the product non-sulfated human natural killer cell-1 (HNK-1) epitope (8).…”