Document Detail

Functional molecular mass of Escherichia coli K92 polysialyltransferase as determined by radiation target analysis.
MedLine Citation:
PMID:  17087504     Owner:  NLM     Status:  MEDLINE    
The polysialyltransferase of Escherichia coli K92 catalyzes the transfer of sialic acid from CMP-sialic acid to a growing chain of polysialic acid at the nonreducing end. The enzyme encoded by the neuS gene is membrane-associated and has been suggested to be organized within a complex of several proteins encoded by the K92 gene cluster. Attempts to prepare a soluble active NeuS enzyme have been unsuccessful. Recent results suggest that de novo synthesis of polysialic acid requires coexpression of four genes from the cluster: neuS, neuE, kpsC, and kpsS. However, elongation of preexisting polysialic acid chains only requires expression of neuS. The molecular organization of the catalytic unit of bacterial polysialyltransferases has not been described. We used radiation inactivation to measure the size of the minimum functional unit catalyzing the polysialyltransferase chain extension and de novo reactions. Membranes harboring NeuS in the presence and absence of other products of the K92 gene cluster were exposed to high-energy electrons. The rate of loss of polysialyltransferase activity reveals the mass of the molecules essential for catalytic activity. We observed that the transfer of neuNAc from CMP-neuNAc to a polysialic acid acceptor is catalyzed by a complex with a target size larger than that of monomeric NeuS. The target size of the unit catalyzing the extension of existing polysialic acid chains does not differ significantly from the size of the unit catalyzing transfer of sialic acid to the endogenous acceptor. Parallel samples of membranes containing NeuS and a green fluorescent protein (GFP) chimera were compared by target analysis. The target size of this structural unit was estimated by analysis of the rate of decay of the GFP-NeuS chimera band migrating in the immunoblots. The target size of the structural unit is larger than expected for a monomer. The results of these experiments show that while the target size of the catalytic activity for K92 polysialyltransferase is larger than a monomer of NeuS, a large complex is not required for catalysis.
Justine Vionnet; Ellis S Kempner; Willie F Vann
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Biochemistry     Volume:  45     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  2006 Nov 
Date Detail:
Created Date:  2006-11-07     Completed Date:  2007-01-19     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  13511-6     Citation Subset:  IM    
Laboratory of Bacterial Polysaccharides, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA.
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MeSH Terms
Blotting, Western
Escherichia coli / enzymology*,  radiation effects
Escherichia coli Proteins / chemistry*,  radiation effects
Models, Biological
Molecular Weight
Sialyltransferases / chemistry*,  radiation effects
Reg. No./Substance:
0/Escherichia coli Proteins; EC 2.4.-/NeuS protein, E coli; EC 2.4.99.-/Sialyltransferases

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