Trans-sialidase activity of Photobacterium damsela alpha2,6-sialyltransferase and its application in the synthesis of sialosides

Abstract

Trans-sialidases catalyze the transfer of a sialic acid from one sialoside to an acceptor to form a new sialoside. alpha2,3-Trans-sialidase activity was initially discovered in the parasitic protozoan Trypanosoma cruzi, and more recently was found in a multifunctional Pasteurella multocida sialyltransferase PmST1. alpha2,8-Trans-sialidase activity was also described for a multifunctional Campylobacter jejuni sialyltransferase CstII. We report here the discovery of the alpha2,6-trans-sialidase activity of a previously reported recombinant truncated bacterial alpha2,6-sialyltransferase from Photobacterium damsela (Delta15Pd2,6ST). This is the first time that the alpha2,6-trans-sialidase activity has ever been identified. Kinetic studies indicate that Delta15Pd2,6ST-catalyzed trans-sialidase reaction follows a ping-pong bi-bi reaction mechanism. Cytidine 5'-monophosphate, the product of sialyltransferase reactions, is not required by the trans-sialidase activity of the enzyme but enhances the trans-sialidase activity modestly as a non-essential activator. Using chemically synthesized Neu5AcalphapNP and LacbetaMU, alpha2,6-linked sialoside Neu5Acalpha2,6LacbetaMU has been obtained in one-step in high yield using the trans-sialidase activity of Delta15Pd2,6ST. In addition to the alpha2,6-trans-sialidase activity, Delta15Pd2,6ST also has alpha2,6-sialidase activity. The multifunctionality is thus a common feature of many bacterial sialyltransferases.

Fig. 1

The pH profiles of the α2,6-sialidase (filled circles) and the α2,6-trans-sialidase activities of Δ15Pd2,6ST obtained by quantitative HPLC analysis. Both Neu5Acα2,6LacβProN₃ (unfilled triangles) and Neu5AcαpNP (unfilled diamonds) were used for the α2,6-trans-sialidase activity assays. 100% conversion was defined as the formation of 1 mM LacβMU for the α2,6-sialidase activity assay or 1 mM Neu5Acα2,6LacβMU for the α2,6-trans-sialidase activity assay.

Fig. 2

Lineweaver–Burk curves for elucidating the kinetic mechanism of the α2,6-trans-sialisase activity of Δ15Pd2,6ST. Double-reciprocal plots of initial velocity were determined for Neu5Acα2,6LacβMU whose concentrations were varied between 1 and 9 mM with the following fixed concentrations of LacβProN₃: (•) 0.5 mM; (□) 1 mM; () 2.5 mM; (○) 5 mM; and (▴) 10 mM. The solid lines corresponded to data fitted to Eq. (1).

Fig. 3

The effects of CMP on the α2,6-sialidase (A) and the α2,6-trans-sialidase (B) activities of Δ15Pd2,6ST. Neu5Acα2,6LacβMU was used for the α2,6-sialidase activity assays. Neu5AcαpNP and LacβMU were used for the α2,6-trans-sialidase activity assays.

Fig. 4

Kinetic mechanisms of the CMP effect on the α2,6-sialidase (A) and the α2,6-trans-sialidase (B) of Δ15Pd2,6ST. Double-reciprocal plots were obtained with Neu5Acα2,6LacβMU (A) or LacβMU (B) as the variable substrates at different fixed concentrations of CMP: () 0 mM; (□) 1 mM; (▴) 10 mM. The solid lines corresponded to data fitted to Eq. (2) (data obtained in the presence of 10 mM CMP did not fit).

Fig. 5

Δ15Pd2,6ST preferentially cleaves α2,6-linked sialic acid. Δ15Pd2,6ST was incubated with biotinylated Neu5Ac-containing α2,3- (A) and (B) α2,6-linked sialyllactosides for 1 h, 4 h, and 13 h, respectively. PmST1, α2,3/α2,6-sialidase from Clostridium perfingens (CP), and α2,3-sialidase from Streptococcus pneumoniae (SP) were used as controls. Abbreviation: N2,3LB, biotinylated α2,3Neu5Ac-linked lactoside; N2,6LB, biotinylated α2,6Neu5Ac-linked lactoside; LB, biotinylated lactoside. Fluorescein-labeled Maackia amurensis Lectin (MAA) (weaker signals were obtained) and fluorescein-labeled Sambucus nigra Lectin (SNA) (stronger signals were obtained) were used to detect α2,3-linked sialyl lactoside and α2,6-linked sialyl lactoside, respectively.

Fig. 6

Alignment of Δ15Pd2,6ST and Δ16PspST6. CMP- and lactose-binding sites are underlined by open triangles and stars, respectively. Amino acid deletions are presented by dashes.

Fig. 7

A general scheme for non-essential activation. Abbreviation: E, enzyme; S, substrate; A, activator; P, product; ES, complex of enzyme and substrate; EA, complex of enzyme and activator; ESA, complex of enzyme, substrate, and activator (Segel 1975).