Research article

Rhodobacter capsulatus porphobilinogen synthase, a high activity metal ion independent hexamer

David W Bollivar¹ , Cheryl Clauson¹ , Rachel Lighthall¹ , Siiri Forbes¹ , Bashkim Kokona² , Robert Fairman² , Lenka Kundrat³ and Eileen K Jaffe³

¹Department of Biology, Illinois Wesleyan University, P.O. Box 2900, Bloomington, IL 61702-2900, USA

²Biology Department, Haverford College, Haverford, PA 19041, USA

³Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA

author email corresponding author email

BMC Biochemistry 2004, 5:17doi:10.1186/1471-2091-5-17

Published:	22 November 2004

Abstract

Background

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.

Results

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 K_m value shows an acidic pK_a of ~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.

Conclusion

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.