Dataset Information

The ATP synthase atpHAGDC (F1) operon from Rhodobacter capsulatus.

ABSTRACT: The atpHAGDC operon of Rhodobacter capsulatus, containing the five genes coding for the F1 sector of the ATP synthase, has been cloned and sequenced. The promoter region has been defined by primer extension analysis. It was not possible to obtain viable cells carrying atp deletions in the R. capsulatus chromosome, indicating that genes coding for ATP synthase are essential, at least under the growth conditions tested. We were able to circumvent this problem by combining gene transfer agent transduction with conjugation. This method represents an easy way to construct strains carrying mutations in indispensable genes.

SUBMITTER: Borghese R

PROVIDER: S-EPMC106900 | biostudies-literature | 1998 Jan

REPOSITORIES: biostudies-literature

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The ATP synthase atpHAGDC (F1) operon from Rhodobacter capsulatus.

Borghese R R Crimi M M Fava L L Melandri B A BA

Journal of bacteriology 19980101 2

The atpHAGDC operon of Rhodobacter capsulatus, containing the five genes coding for the F1 sector of the ATP synthase, has been cloned and sequenced. The promoter region has been defined by primer extension analysis. It was not possible to obtain viable cells carrying atp deletions in the R. capsulatus chromosome, indicating that genes coding for ATP synthase are essential, at least under the growth conditions tested. We were able to circumvent this problem by combining gene transfer agent trans ...[more]

PMID: 9440534

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Project description:The enzyme porphobilinogen synthase (PBGS), which is central to the biosynthesis of heme, chlorophyll and cobalamins, has long been known to use a variety of metal ions and has recently been shown able to exist in two very different quaternary forms that are related to metal ion usage. This paper reports new information on the metal ion independence and quaternary structure of PBGS from the photosynthetic bacterium Rhodobacter capsulatus.The gene for R. capsulatus PBGS was amplified from genomic DNA and sequencing revealed errors in the sequence database. R. capsulatus PBGS was heterologously expressed in E. coli and purified to homogeneity. Analysis of an unusual phylogenetic variation in metal ion usage by PBGS enzymes predicts that R. capsulatus PBGS does not utilize metal ions such as Zn2+, or Mg2+, which have been shown to act in other PBGS at either catalytic or allosteric sites. Studies with these ions and chelators confirm the predictions. A broad pH optimum was determined to be independent of monovalent cations, approximately 8.5, and the Km value shows an acidic pKa of approximately 6. Because the metal ions of other PBGS affect the quaternary structure, gel permeation chromatography and analytical ultracentrifugation experiments were performed to examine the quaternary structure of metal ion independent R. capsulatus PBGS. The enzyme was found to be predominantly hexameric, in contrast with most other PBGS, which are octameric. A protein concentration dependence to the specific activity suggests that the hexameric R. capsulatus PBGS is very active and can dissociate to smaller, less active, species. A homology model of hexameric R. capsulatus PBGS is presented and discussed.The evidence presented in this paper supports the unusual position of the R. capsulatus PBGS as not requiring any metal ions for function. Unlike other wild-type PBGS, the R. capsulatus protein is a hexamer with an unusually high specific activity when compared to other octameric PBGS proteins.

Dataset Information

The ATP synthase atpHAGDC (F1) operon from Rhodobacter capsulatus.

Publications

The ATP synthase atpHAGDC (F1) operon from Rhodobacter capsulatus.

Similar Datasets

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