Premium
Flexibility of Substrate Binding of Cytosine‐5′‐Monophosphate‐ N ‐Acetylneuraminate Synthetase (CMP‐Sialate Synthetase) from Neisseria meningitidis : An Enabling Catalyst for the Synthesis of Neo‐sialoconjugates
Author(s) -
He Ning,
Yi Dong,
Fessner WolfDieter
Publication year - 2011
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.201100412
Subject(s) - chemistry , stereochemistry , cytosine , substrate (aquarium) , active site , sialic acid , enzyme , biochemistry , dna , oceanography , geology
We have successfully established a simple continuous colorimetric assay for the sensitive and reliable quantification of cytosine‐5′‐monophosphate‐acetylneuraminate synthetase (CMP‐sialate synthetase, CSS) activity based on the pH change of a released proton equivalent upon nucleotide activation of Neu5Ac or related analogues. Using this method, steady‐state kinetic data of Neisseria meningitidis CSS were determined for cytosine‐5′‐triphosphate (CTP), N ‐acetylneuraminic acid (Neu5Ac) and eleven structural variations of the sialic acid, including backbone truncation, deamination, epimerization, and several N ‐acyl modifications. These data further demonstrate the unusual versatility of the N. meningitidis CSS for a general access to sialoconjugates containing non‐natural sialic acid analogues. Remarkably, the assay allows covering a broad range of substrate parameters that span over more than three orders of magnitude for K M and k cat measurements. With the aid of a structural model built from X‐ray crystal structure data, the kinetic data could be used to interpret potential protein contributions in substrate binding of Neu5Ac and its analogues. The Neu5Ac analogues were used for the preparation of neo‐sialoconjugate analogues of 2,6‐sialyllactose in a one‐pot two‐enzyme cascade, using N. meningitidis CSS together with the novel α2,6‐sialyltransferase (α2,6SiaT) from Photobacterium leiognathi , which proved to be highly effective because of its optimum activity at alkaline pH, appropriately matching the requirements from the overall reaction system.