A C-Terminal Phosphatase Module Conserved in Vertebrate CMP-Sialic Acid Synthetases Provides a Tetramerization Interface for the Physiologically Active Enzyme |
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Authors: | Melanie Oschlies,Thomas Haselhorst,Katharina Stummeyer,Birgit Weinhold,Mark von Itzstein,Anja-K. Mü nster-Kü hnel |
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Affiliation: | 1 Institut für Zelluläre Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany 2 Institut für Mikrobiologie und Genetik, Abteilung Molekulare Strukturbiologie, Georg-August-Universität, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany 3 Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland 4222, Australia |
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Abstract: | The biosynthesis of sialic acid-containing glycoconjugates is crucial for the development of vertebrate life. Cytidine monophosphate-sialic acid synthetase (CSS) catalyzes the metabolic activation of sialic acids. In vertebrates, the enzyme is chimeric, with the N-terminal domain harboring the synthetase activity. The function of the highly conserved C-terminal domain (CSS-CT) is unknown. To shed light on its biological function, we solved the X-ray structure of murine CSS-CT to 1.9 Å resolution. CSS-CT is a stable shamrock-like tetramer that superimposes well with phosphatases of the haloacid dehalogenase superfamily. However, a region found exclusively in vertebrate CSS-CT appears to block the active-site entrance. Accordingly, no phosphatase activity was observed in vitro, which points toward a nonenzymatic function of CSS-CT. A computational three-dimensional model of full-length CSS, in combination with in vitro oligomerization studies, provides evidence that CSS-CT serves as a platform for the quaternary organization governing the kinetic properties of the physiologically active enzyme as demonstrated in kinetic studies. |
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Keywords: | CIP, calf intestine phosphatase CMP, cytidine monophosphate CSS, CMP-sialic acid synthetase CT, C-terminal domain HAD, haloacid dehalogenase KDN, 2-keto-3-deoxy- smallcaps" >d-glycero- smallcaps" >d-galacto-nononic acid KDO, 3-deoxy- smallcaps" >d-manno-octulosonic acid MAD, multiple-wavelength anomalous dispersion ManNAc-6P, N-acetylmannosamine-6-phosphate mCSS-NT, N-terminal domain of mouse CSS Neu5Ac, N-acetylneuraminic acid NPS, Neu5Ac9P synthetase NT, N-terminal domain PC, pyruvate carboxylase pNPP, p-nitrophenylphosphate STD, saturation transfer difference SEC, size exclusion chromatography SeMet, selenomethionine Sia, sialic acid |
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