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Identification of yacE (coaE) as the structural gene for dephosphocoenzyme A kinase in Escherichia coli K-12.

ABSTRACT: Dephosphocoenzyme A (dephospho-CoA) kinase catalyzes the final step in coenzyme A biosynthesis, the phosphorylation of the 3'-hydroxy group of the ribose sugar moiety. Wild-type dephospho-CoA kinase from Corynebacterium ammoniagenes was purified to homogeneity and subjected to N-terminal sequence analysis. A BLAST search identified a gene from Escherichia coli previously designated yacE encoding a highly homologous protein. Amplification of the gene and overexpression yielded recombinant dephospho-CoA kinase as a 22.6-kDa monomer. Enzyme assay and nuclear magnetic resonance analyses of the product demonstrated that the recombinant enzyme is indeed dephospho-CoA kinase. The activities with adenosine, AMP, and adenosine phosphosulfate were 4 to 8% of the activity with dephospho-CoA. Homologues of the E. coli dephospho-CoA kinase were identified in a diverse range of organisms.

SUBMITTER: Mishra P

PROVIDER: S-EPMC99492 | biostudies-literature | 2001 May

REPOSITORIES: biostudies-literature

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Identification of yacE (coaE) as the structural gene for dephosphocoenzyme A kinase in Escherichia coli K-12.

Mishra P P Park P K PK Drueckhammer D G DG

Journal of bacteriology 20010501 9

Dephosphocoenzyme A (dephospho-CoA) kinase catalyzes the final step in coenzyme A biosynthesis, the phosphorylation of the 3'-hydroxy group of the ribose sugar moiety. Wild-type dephospho-CoA kinase from Corynebacterium ammoniagenes was purified to homogeneity and subjected to N-terminal sequence analysis. A BLAST search identified a gene from Escherichia coli previously designated yacE encoding a highly homologous protein. Amplification of the gene and overexpression yielded recombinant dephosp ...[more]

PMID: 11292795

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Project description:The polysaccharide chains of enterobacterial common antigen (ECA) are comprised of the trisaccharide repeat unit Fuc4NAc-ManNAcA-GlcNAc, where Fuc4NAc is 4-acetamido-4,6-dideoxy-D-galactose, ManNAcA is N-acetyl-D-mannosaminuronic acid, and GlcNAc is N-acetyl-D-glucosamine. Individual trisaccharide repeat units are assembled as undecaprenyl-linked intermediates in a sequence of reactions that culminate in the transfer of Fuc4NAc from TDP-Fuc4NAc to ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid II) to yield Fuc4NAc-ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid III), the donor of trisaccharide repeat units for ECA polysaccharide chain elongation. Most of the genes known to be involved in ECA assembly are located in the wec gene cluster located at ca. 85.4 min on the Escherichia coli chromosome. The available data suggest that the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase also resides in the wec gene cluster; however, the location of this gene has not been unequivocally defined. Previous characterization of the nucleotide sequence of the wec gene cluster in the region between o416 and wecG revealed that it contained three open reading frames: o74, o204, and o450. In contrast, the results of experiments described in the current investigation revealed that it contains only two open reading frames, o359 and o450. Mutants of E. coli possessing null mutations in o359 were unable to synthesize ECA, and they accumulated lipid II. In addition, the in vitro incorporation of [(3)H]FucNAc from TDP-[(3)H]Fuc4NAc into lipid II was not observed in reaction mixtures using cell extracts obtained from these mutants as a source of enzyme. The ECA-negative phenotype of these mutants was complemented by plasmid constructs containing the wild-type o359 allele, and Fuc4NAc transferase activity was demonstrated by using cell extracts obtained from the complemented mutants. Furthermore, partially purified o359 gene product, expressed as recombinant C-terminal His-tagged protein, was able to catalyze the in vitro transfer of [(3)H]Fuc4NAc from TDP-[(3)H]Fuc4NAc to lipid II. Our data support the conclusion that o359 of the wec gene cluster of E. coli is the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase involved in the synthesis ECA trisaccharide repeat units.

Dataset Information

Identification of yacE (coaE) as the structural gene for dephosphocoenzyme A kinase in Escherichia coli K-12.

Publications

Identification of yacE (coaE) as the structural gene for dephosphocoenzyme A kinase in Escherichia coli K-12.

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