DOI: 10.1002/adsc.201100412 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 Ning He, a Dong Yi, a and Wolf-Dieter Fessner a, * a Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Petersenstrasse 22, 64287 Darmstadt, Germany Fax: (+ 49)-6151-16-6636; e-mail: fessner@tu-darmstadt.de Received: May 20, 2011; Revised: July 26, 2011; Published online: August 30, 2011 Dedicated to Prof. Horst Prinzbach on the occasion of his 80 th birthday. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adcs.201100412. Abstract: We have successfully established a simple continuous colorimetric assay for the sensitive and reliable quantification of cytosine-5’-monophos- phate-acetylneuraminate synthetase (CMP-sialate synthetase, CSS) activity based on the pH change of a released proton equivalent upon nucleotide activa- tion of Neu5Ac or related analogues. Using this method, steady-state kinetic data of Neisseria menin- gitidis CSS were determined for cytosine-5’-triphos- phate (CTP), N-acetylneuraminic acid (Neu5Ac) and eleven structural variations of the sialic acid, includ- ing backbone truncation, deamination, epimeriza- tion, and several N-acyl modifications. These data further demonstrate the unusual versatility of the N. meningitidis CSS for a general access to sialoconju- gates 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 measure- ments. 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 prepara- tion of neo-sialoconjugate analogues of 2,6-sialyllac- tose in a one-pot two-enzyme cascade, using N. men- ingitidis CSS together with the novel a2,6-sialyltrans- ferase (a2,6SiaT) from Photobacterium leiognathi, which proved to be highly effective because of its op- timum activity at alkaline pH, appropriately match- ing the requirements from the overall reaction system. Keywords: enzyme catalysis; glycoconjugates; glyco- sylation; oligosaccharides; sialic acids Introduction Sialic acids are a family of acidic 9-carbon sugars that represent one of the most important constituents of cell-surface glycoconjugates in biological systems. [1,2] The predominant member of the family is 5-acetami- do-d-glycero-d-galacto-2-nonulosonic acid (N-acetyl- neuraminic acid, Neu5Ac, 1; Scheme 1), among more than 50 natural derivatives that have been identified so far, including the non-aminated derivative 3-deoxy- d-glycero-d-galacto-2-nonulosonic acid (KDN, 2). [3] Natural sialic acid derivatives typically incorporate various types of N- or O-modification by acetylation, Scheme 1. Biosynthesis of sialoconjugates along the Leloir pathway. 2384  2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Synth. Catal. 2011, 353, 2384 – 2398 FULL PAPERS