A conserved cysteine residue in yeast uroporphyrinogen decarboxylase is not essential for enzymatic activity.

Uroporphyrinogen decarboxylase catalyzes the fifth step of heme biosynthesis in Saccharomyces cerevisiae. Studies utilizing sulfhydryl-specific reagents suggest that the enzyme requires a cysteine residue within the catalytic site. This hypothesis was tested directly by site-directed mutagenesis of highly conserved cysteine-52 to serine or alanine. Plasmids containing these mutations were able to complement a hem6 mutant strain. In addition, properties associated with decreased uroporphyrinogen decarboxylase activity were not detected in the mutant strain transformed with these mutant plasmids. These results suggest that the conserved cysteine-52 by itself is not essential for enzymatic activity.

The HMG domain of the ROX1 protein mediates repression of HEM13 through overlapping DNA binding and oligomerization functions.

The ROX1 gene of Saccharomyces cerevisiae encodes a protein required for the repression of genes expressed under anaerobic conditions. ROX1 belongs to a family of DNA binding proteins which contain the high mobility group motif (HMG domain). To ascertain whether the HMG domain of ROX1 is required for specific DNA binding we synthesized a series of ROX1 protein derivatives, either in vitro or in Escherichia coli as fusions to glutathione S-transferase (GST) protein, and tested them for their ability to bind to DNA. Both ROX1 proteins that were synthesized in vitro and GST-ROX1 fusion proteins containing the intact HMG domain were able to bind to specific target DNA sequences. In contrast, ROX1 proteins which contained deletions within the HMG domain were no longer capable of binding to DNA. The oligomerization of ROX1 in vitro was demonstrated using affinity-purified GST-ROXI protein and ROX1 labelled with [35S]methionine. Using various ROX1 protein derivatives we were able to demonstrate that the domain required for ROX1-ROX1 interaction resides within the N-terminal 100 amino acids which constitute the HMG domain. Therefore, the HMG domain is required for both DNA binding activity and oligomerization of ROX1.