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Modulating the regioselectivity of a Pasteurella multocida sialyltransferase for biocatalytic production of 3′- and 6′-sialyllactose

Paper ID Volume ID Publish Year Pages File Format Full-Text
16849 42617 2015 9 PDF Available
Title
Modulating the regioselectivity of a Pasteurella multocida sialyltransferase for biocatalytic production of 3′- and 6′-sialyllactose
Abstract

•Sialyltransferase mutations change regioselectivity in trans-sialidase reaction.•P34H changes an α-2,3- to an α-2,6-sialyltransferase.•P34H preferentially yields 6′-sialyllactose in trans-sialidase reactions.•E271F and R313Y preferentially yield 3′-sialyllactose in trans-sialidase reactions.

Several bacterial sialyltransferases have been reported to be multifunctional also catalysing sialidase and trans-sialidase reactions. In this study, we examined the trans-sialylation efficacy and regioselectivity of mutants of the multifunctional Pasteurella multocida sialyltransferase (PmST) for catalysing the synthesis of 3′- and 6′-sialyllactose using casein glycomacropeptide as sialyl-donor and lactose as acceptor. The mutation P34H led to a 980-fold increase in α-2,6-sialyltransferase activity (with cytidine-5′-monophospho-N-acetylneuraminic acid as donor), while its α-2,3-sialyltransferase activity was abolished. Histidine in this position is conserved in α-2,6-sialyltransferases and has been suggested, and recently confirmed, to be the determinant for strict regiospecificity in the sialyltransferase reaction. Our data verified this theorem. In trans-sialidase reactions, the P34H mutant displayed a distinct preference for 6′-sialyllactose synthesis but low levels of 3′-sialyllactose were also produced. The sialyllactose yield was however lower than when using PmSTWT under optimal conditions for 6′-sialyllactose formation. The discrepancy in regiospecificity between the two reactions could indicate subtle differences in the substrate binding site in the two reactions. In contrast, the two mutations E271F and R313Y led to preferential synthesis of 3′-sialyllactose over 6′-sialyllactose and the double mutant (PmSTE271F/R313Y) exhibited the highest α-2,3-regioselectivity via reduced sialidase and α-2,6-trans-sialidase activity. The double mutant PmSTE271F/R313Y thus showed the highest α-2,3-regioselectivity and constitutes an interesting enzyme for regioselective synthesis of α-2,3-sialylated glycans. This study has expanded the understanding of the structure-function relationship of multifunctional, bacterial sialyltransferases and provided new enzymes for regioselective glycan sialylation.

Keywords
cGMP, casein glycomacropeptide; CMP-, cytidine-5′-monophospho-N-acetylneuraminic acid; GOS, galactooligosaccharides; HPAEC-PAD, high-performance anion exchange chromatography with pulsed amperometric detection; N-His6-tag, N-terminal His6-tag; PmST, Paste
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Modulating the regioselectivity of a Pasteurella multocida sialyltransferase for biocatalytic production of 3′- and 6′-sialyllactose
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 78, October 2015, Pages 54–62
Authors
, , , , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us