EXPRESSION, PURIFICATION AND CHARACTERIZATION OF THE PRODUCT FROM THEBACILLUS-SUBTILIS HEMD GENE, UROPORPHYRINOGEN-III SYNTHASE

Uroporphyrinogen III synthase, the product of the hemD gene, is the en zyme responsible for the cyclisation of the linear tetrapyrrole, hydro xymethylbilane. The hemD gene isolated from Bacillus subtilis was mani pulated by PCR to enable direct cloning behind a synthetic ribosome-bi nding site downstream of tandem bacteriophage lambda P-R and P-L promo ters in a pCE30-derived vector. Following thermal induction of transcr iption, the resulting plasmid (pPS21) directed the synthesis of uropor phyrinogen III synthase. The protein produced was soluble and was read ily purified. Pure uroporphyrinogen III synthase is monomeric with an isoelectric point of 4.1 and an optimum pH for activity of 8.3. Its sp ecific activity by assay using synthetic hydroxymethylbilane as substr ate is 565 units mg(-1) and the K-m for this substrate is 330 +/- 30 n M. The N-terminal sequence of the enzyme is Met-Glu-Asn-Asp-Phe-Pro-Le u, in agreement with the gene-derived sequence. Studies based on amino acid modifications suggest that arginine, lysine and probably histidi ne residues are essential for the activity of uroporphyrinogen III syn thase. Significantly, this synthase from B. subtilis is substantially more thermostable than the enzymes from previously studied sources.