Expression, Purification and Characterisation of the Product from the Bacillus Subtilis hemD gene, Uroporphyrinogen III Synthase

Uroporphyrinogen III synthase, the product of the hemD gene, is the enzyme responsible for the cyclisation of the linear tetrapyrrole, hydroxymethylbilane. The hemD gene isolated from Bacillus subtilis was manipulated by PCR to enable direct cloning behind a synthetic ribosome-binding site downstream of tandem bacteriophage lambda PR and PL promoters in a pCE30-derived vector. Following thermal induction of transcription, the resulting plasmid (pPS21) directed the synthesis of uroporphyrinogen III synthase. The protein produced was soluble and was readily purified. Pure uroporphyrinogen III synthase is monomeric with an isoelectric point of 4.1 and an optimum pH for activity of 8.3. Its specific activity by assay using synthetic hydroxymethylbilane as substrate is 565 units mg-1 and the Km for this substrate is 330 +/- 30 nM. The N-terminal sequence of the enzyme is Met-Glu-Asn-Asp-Phe-Pro-Leu, in agreement with the gene-derived sequence. Studies based on amino acid modifications suggest that arginine, lysine and probably histidine residues are essential for the activity of uroporphyrinogen III synthase. Significantly, this synthase from B. subtilis is substantially more thermostable than the enzymes from previously studied sources.