Journal Article

Molecular cloning of a unique CMP–sialic acid synthetase that effectively utilizes both deaminoneuraminic acid (KDN) and <i>N</i>-acetylneuraminic acid (Neu5Ac) as substrates

Daisuke Nakata, Anja-K. Münster, Rita Gerardy-Schahn, Naohito Aoki, Tsukasa Matsuda and Ken Kitajima

in Glycobiology

Published on behalf of Society for Glycobiology

Volume 11, issue 8, pages 685-692
Published in print August 2001 | ISSN: 0959-6658
Published online August 2001 | e-ISSN: 1460-2423 | DOI: http://dx.doi.org/10.1093/glycob/11.8.685
Molecular cloning of a unique CMP–sialic acid synthetase that effectively utilizes both deaminoneuraminic acid (KDN) and N-acetylneuraminic acid (Neu5Ac) as substrates

Show Summary Details

Preview

2-Keto-3-deoxy-d-glycero-d-galacto-nononic acid (KDN) is a sialic acid (Sia) that is ubiquitously expressed in vertebrates during normal development and tumorigenesis. Its expression is thought to be regulated by multiple biosynthetic steps catalyzed by several enzymes, including CMP-Sia synthetase. Using crude enzyme preparations, it was shown that mammalian CMP-Sia synthetases had very low activity to synthesize CMP-KDN from KDN and CTP, and the corresponding enzyme from rainbow trout testis had high activity to synthesize both CMP-KDN and CMP-N-acetylneuraminic acid (Neu5Ac) (Terada et al. [1993] J. Biol. Chem., 268, 2640–2648). To demonstrate if the unique substrate specificity found in the crude trout enzyme is conveyed by a single enzyme, cDNA cloning of trout CMP-Sia synthetase was carried out by PCR-based strategy. The trout enzyme was shown to consist of 432 amino acids with two potential nuclear localization signals, and the cDNA sequence displayed 53.8% identity to that of the murine enzyme. Based on the Vmax/Km values, the recombinant trout enzyme had high activity toward both KDN and Neu5Ac (1.1 versus 0.68 min–1). In contrast, the recombinant murine enzyme had 15 times lower activity toward KDN than Neu5Ac (0.23 versus 3.5 min–1). Northern blot analysis suggested that several sizes of the mRNA are expressed in testis, ovary, and liver in a tissue-specific manner. These results indicate that at least one cloned enzyme has the ability to utilize both KDN and Neu5Ac as substrates efficiently and is useful for the production of CMP-KDN.

Keywords: Key words: sialic acid/deaminoneuraminic acid/CMP-KDN synthetase/CMP-Neu5Ac synthetase/CMP–sialic acid synthetase

Journal Article.  6315 words.  Illustrated.

Subjects: Carbohydrates

Full text: subscription required

How to subscribe Recommend to my Librarian

Users without a subscription are not able to see the full content. Please, subscribe or login to access all content.