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Applied Microbiology and Biotechnology

, Volume 93, Issue 6, pp 2411–2423 | Cite as

Pasteurella multocida CMP-sialic acid synthetase and mutants of Neisseria meningitidis CMP-sialic acid synthetase with improved substrate promiscuity

  • Yanhong Li
  • Hai Yu
  • Hongzhi Cao
  • Saddam Muthana
  • Xi ChenEmail author
Biotechnologically relevant enzymes and proteins

Abstract

Cytidine 5′-monophosphate (CMP)-sialic acid synthetases (CSSs) catalyze the formation of CMP-sialic acid from CTP and sialic acid, a key step for sialyltransferase-catalyzed biosynthesis of sialic acid-containing oligosaccharides and glycoconjugates. More than 50 different sialic acid forms have been identified in nature. To facilitate the enzymatic synthesis of sialosides with diverse naturally occurring sialic acid forms and their non-natural derivatives, CMP-sialic acid synthetases with promiscuous substrate specificity are needed. Herein we report the cloning, characterization, and substrate specificity studies of a new CSS from Pasteurella multocida strain P-1059 (PmCSS) and a CSS from Haemophillus ducreyi (HdCSS). Based on protein sequence alignment and substrate specificity studies of these two CSSs and a Neisseria meningitidis CSS (NmCSS), as well as crystal structure modeling and analysis of NmCSS, NmCSS mutants (NmCSS_S81R and NmCSS_Q163A) with improved substrate promiscuity were generated. The strategy of combining substrate specificity studies of enzymes from different sources and protein crystal structure studies can be a general approach for designing enzyme mutants with improved activity and substrate promiscuity.

Keywords

Carbohydrate synthesis CMP-sialic acid CMP-sialic acid synthetase Mutagenesis Sialic acid Substrate specificity 

Notes

Acknowledgments

This work was support by NIH grants R01GM076360 and R01HD065122, the Alfred P. Sloan Research Fellowship, the Camille Dreyfus Teacher-Scholarship, and the UC-Davis Chancellor’s Fellowship.

Supplementary material

253_2011_3579_MOESM1_ESM.doc (88 kb)
ESM 1 (DOC 88 kb)

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Yanhong Li
    • 1
  • Hai Yu
    • 1
  • Hongzhi Cao
    • 1
    • 2
  • Saddam Muthana
    • 1
    • 3
  • Xi Chen
    • 1
    Email author
  1. 1.Department of ChemistryUniversity of California-DavisDavisUSA
  2. 2.National Glycoengineering Research CenterShandong UniversityJinanChina
  3. 3.Chemical Biology Laboratory, National Cancer Institute at FrederickNational Institutes of HealthFrederickUSA

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