Active‐Site His85 of Pasteurella dagmatis Sialyltransferase Facilitates Productive Sialyl Transfer and So Prevents Futile Hydrolysis of CMP‐Neu5Ac

Abstract

Sialyltransferases of the GT-80 glycosyltransferase family are considered multifunctional because of the array of activities detected. They exhibit glycosyl transfer, trans-sialylation, and hydrolysis activities. How these enzymes utilize their active-site residues in balancing the different enzymatic activities is not well understood. In this study of Pasteurella dagmatis α2,3sialyltransferase, we show that the conserved His85 controls efficiency and selectivity of the sialyl transfer. A His85→Asn variant was 200 times less efficient than wild-type for sialylation of lactose, and exhibited relaxed site selectivity to form not only the α2,3- but also the α2,6-sialylated product (21 %). The H85N variant was virtually inactive in trans-sialylation but showed almost the same CMP-Neu5Ac hydrolase activity as wild-type. The competition between sialyl transfer and hydrolysis in the conversion of CMP-Neu5Ac was dependent on the lactose concentration; this was characterized by a kinetic partition ratio of 85 m for the H85N variant, compared to 17 000 m for the wild-type enzyme. His85 promotes the productive sialyl transfer to lactose and so prevents hydrolysis of CMP-Neu5Ac in the reaction.

Leloir Glycosyltransferases in Applied Biocatalysis: A Multidisciplinary Approach

“…The high glycosylation efficiency with Leloir GTs arises from a favorable thermodynamic equilibrium K eq in these examples, determined by the sugar nucleotide donor and carbohydrate or aglycone acceptor, pH, and ionic strength. As mentioned earlier, in a few examples the hydrolysis of the NDP-sugar donor has been reported for Leloir GTs [70,71,72,73,74,75,76,77]. In these particular cases, it is important to emphasize that the glycosylation with Leloir GTs is under kinetic control, and the sugar acceptor and water are competing nucleophiles throughout the entire course of reaction [70,71,72,73,74,75,76,77].…”

“…For instance, the bacterial sialyltransferase from Pasteurella dagmatis hydrolyzed the rather hydrolysis-prone CMP-Neu5Ac in the absence of another substrate [74]. Directed evolution has been shown to be an effective tool to diminish the degree of hydrolysis of NDP sialyl donor [75,76]. In comparison to hemiketals, hemiacetals are more stable sugar donor nucleotides (i.e., GDP- l -fucose).…”

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

“…There have been various attempts to control hydrolytic sidereactivity of the 2,3SiaT and 2,6SiaT from Pasteurella multocida, [33] Photobacterium sp. [34,35] and P. phosphoreum, [36] Pasteurella dagmatis, [37,38] or Photobacterium damselae, [25] by simple addition of organic solvents, pH adjustment, or rational active-site-directed mutagenesis. Unfortunately,m ost efforts in suppressing side reactions by site-directed mutagenesis were accompanied by drastically decreased transfer rates.…”

“…Unfortunately,m ost efforts in suppressing side reactions by site-directed mutagenesis were accompanied by drastically decreased transfer rates. [33,38] The highly polar nature and large molecular sizes of both the CMP-Sia-andt he Sia-accepting glycan substrates, which have to be juxtaposed in the transition state for regiospecific sialyl transfer,r equirea n intricateh ydrogen-bonding network to protein residues in the active site, as evident from severalX -ray crystal structures of GT80 enzymes with bound substrate analogues. In order to address this specific situation for the 2,3SiaT pph ,w ed ecided to embarko nadirected evolution study involving an extended set of 13 amino acid residues in the active site that were either known or presumed to be in close contact with substrates during the catalytic transfer event.…”

“…Most methods for assaying sialyltransferase activity reported so far are discontinuous and involve laborious quenching of the catalytic reaction by acido rh eating followed by product analysis. [36,38,42] HPLC analysiso ff luorescently labeled substrates [33] is indirect, and surrogate substrates may show ak inetic behavior different from the native substrates. [43] The only continuous assay for SiaT activity is based on the indirect measurement of CMP release by an enzymatic link to NADPH consumption, which is inappropriate for discrimination of sialidase activity.…”