Loss-of-Function Mutation in Bi-Functional Marine Bacterial Sialyltransferase

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

“…To our dismay,w ec ouldn ot confirm the literatured ata reported for variant E342A,w hich wasc laimed to have approximately five times higher k cat for CMP-Neu5Ac and nine times higher k cat for lactose. [36] Conversely,u nder our assay conditions we detected about 40 %r educed k cat values and a4 0-fold lower binding affinity towards CMP-Neu5Ac (Table S5). Thel atter is plausible because the mutation concerns ah ighly conserved residue of the CMP-binding motif in GT80 sialyltransferases.…”

“…It was claimed that residueE 342 in 2,3SiaT pph is important for sialidase activity (path C), because the mutationE 342A abolished a2,3-sialyllactose hydrolysis. [36] Kinetic data for the selected combination variants indeed did not show furtheri mprovement compared to the parent A151D,L 387A,o rS 350T/S360Tv ariants. The main similarity of the combined variants is the significant reduction of k cat values for sialyltransfer activity.E ven am ajor reduction in hydrolytic efficiency for the combinedA 151D/L387A mutation is not useful for practical applications because the low sialyltransfer activity would cause ah igh stationaryc oncentration of CMP- Table 2.…”

“…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.…”

“…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.…”

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

“…All these residues directly interact with the substrate in the acceptor‐binding pocket or are located close to it (Figure ). Additionally, for an α2,3‐sialyltransferase from Photobacterium phosphoreum it was shown that Glu342 (Glu338 in PmST1) in the donor‐binding pocket was important for sialidase activity …”