Chemoenzymatic synthesis of CMP-sialic acid derivatives by a one-pot two-enzyme system: Comparison of substrate flexibility of three microbial CMP-sialic acid synthetases

Hai Yu, Hui Yu, Rebekah Karpel, Xi Chen

Research output: Contribution to journalArticlepeer-review

150 Scopus citations

Abstract

Three microbial CMP-sialic acid synthetases were cloned from Neisseria meningitidis, Streptococcus agalactiae, and Escherichia coli, respectively. Their activities in the production of CMP-sialic acid analogs were compared by HPLC analysis. The N. meningitidis synthetase was used in the preparative synthesis of eight CMP-sialic acid derivatives in a one-pot two-enzyme system. Three C terminal His 6-tagged recombinant microbial CMP-sialic acid synthetases [EC 2.7.7.43] cloned from Neisseria meningitidis group B, Streptococcus agalactiae serotype V, and Escherichia coli K1, respectively, were evaluated for their ability in the synthesis of CMP-sialic acid derivatives in a one-pot two-enzyme system. In this system, N-acetylmannosamine or mannose analogs were condensed with pyruvate, catalyzed by a recombinant sialic acid aldolase [EC 4.1.3.3] cloned from E. coli K12 to provide sialic acid analogs as substrates for the CMP-sialic acid synthetases. The substrate flexibility and the reaction efficiency of the three recombinant CMP-sialic acid synthetases were compared, first by qualitative screening using thin layer chromatography, and then by quantitative analysis using high performance liquid chromatography. The N. meningitidis synthetase was shown to have the highest expression level, the most flexible substrate specificity, and the highest catalytic efficiency among the three synthetases. Finally, eight sugar nucleotides, including cytidine 5′-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) and its derivatives with substitutions at carbon-5, carbon-8, or carbon-9 of Neu5Ac, were synthesized in a preparative (100-200 mg) scale from their 5- or 6-carbon sugar precursors using the N. meningitidis synthetase and the aldolase.

Original languageEnglish (US)
Pages (from-to)6427-6435
Number of pages9
JournalBioorganic and Medicinal Chemistry
Volume12
Issue number24
DOIs
StatePublished - Dec 15 2004

Keywords

  • Aldolase
  • CMP-sialic acid
  • CMP-sialic acid synthetase
  • Enzymatic synthesis

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Organic Chemistry
  • Drug Discovery
  • Pharmaceutical Science

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