Abstract

The enzyme porphobilinogen synthase (PBGS) catalyzes the conversion of two molecules of δ-aminolevulinic acid (ALA) into porphobilinogen (PBG) in the first common step of the tetrapyrrole biosynthesis pathway. A homology model of the R. sphaeroides PBGS was created by Dr. Eileen K. Jaffe at Fox Chase Cancer Center based on comparison to the crystal structure of Pseudomonas aeruginosa. The proposed structure suggests that there are four cysteines in close proximity to the active site. Three of these cysteines are not present in the highly similar R. capsulatus sequence. Under oxidizing conditions these residues can potentially participate in the formation of disulfide bonds, which would be potential targets for reducing agents, such as β-mercaptoethanol. This is supported by the fact that a number of PBGS enzymes require the addition of β-mercaptoethanol for full activity, including the Rhodobacter sphaeroides enzyme. To test if the three cysteines that are present in the R. sphaeroides enzyme, and not that of R. capsulatus, are responsible for the dependence of R. sphaeroides on a reducing agent (β-mercaptoethanol), site-directed mutagenesis was performed on R. sphaeroides. After the presence of the mutations was confirmed through sequencing, the altered enzymes were isolated and tested for β-mercaptoethanol sensitivity. A decrease in sensitivity is found in all three mutants when compared to wild type. In two mutants of these mutants, C103D and C265V, the difference is very pronounced. A larger goal was to test whether the current structural model is sufficient to serve as a guide for further work on the structure-function relationships in the R. sphaeroides PBGS. Our results indicate that the system is more complicated than anticipated. Further study is needed to clarify the role of cysteine residues.

Disciplines

Biochemistry



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