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

Abstract

Uroporphyrinogen I11 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 A P, and PL promoters in a pCE30-derived vector. Following thermal induction of transcription, the resulting plasmid (pPS21) directed the synthesis of uroporphyrinogen 111 synthase. The protein produced was soluble and was readily purified. Pure uroporphyrinogen I11 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-' and the K , for this substrate is 330 2 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 111 synthase. Significantly, this synthase from B. subtilis is substantially more thermostable than the enzymes from previously studied sources.

The pleiotropic transcriptional regulator NlpR contributes to the modulation of nitrogen metabolism, lipogenesis and triacylglycerol accumulation in oleaginous rhodococci

“…The putative transcriptional regulator NlpR is coded by the RHA1_RS31140 (formerly ro06368 ) and opag_03371 (PD630_LPD03034) genes in R. jostii RHA1 and R. opacus PD630 respectively. The predicted NlpR proteins possess two conserved domains: The N‐terminal region containing an enzyme‐like domain similar to uroporphyrinogen‐III synthases (HemD), which catalyzes the uroporphyrinogen III synthesis from hydroxymethylbilane and as part of the pyrrole‐containing compounds biosynthesis (Stamford et al ., ), and the C‐terminal region similar to the DNA binding domains of OmpR‐like response regulators. Full protein sequence alignment by BLAST algorithm with all bacterial proteins available in database, showed the occurrence of NlpR homologs in all analyzed Rhodococcus species as well as in other actinobacteria belonging to Nocardia , Gordonia , Mycobacterium , Streptomyces and Amycolatopsis genera, with significant coverage percentages (> 95%) and identities (I > 50%) (Fig.…”

Global probabilistic annotation of metabolic networks enables enzyme discovery

“…One example is the B. subtilis gene hemD , known to be responsible for the uroporphyrinogen-III synthase activity 37 (EC 4.2.1.75). The sequence identity of hemD to the closest Swiss-Prot sequence performing its correct function is only ~24%; however, GLOBUS assigned a high probability (P=0.86) to the correct EC number because of the excellent context associations with its neighboring enzymes at this location: the gene clustering Z-score (defined as the number of standard deviations from the mean based on all gene-gene context scores, see Methods) is 21.2, the co-expression Z-score is 5.64.…”

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product

“…The advent of recombinant-DNA technology allowed the enzyme to be produced in much larger quantities (139,140), and the development of a fluorescencebased assay (141) coupled with more accurate high-performance liquid chromatography (HPLC) analysis to separate the type I and III isomers made the assays much more rapid and accurate (142). Consequently, after the initial laborious methods to isolate the first purified UroS enzyme from human blood (143), quite a few UroSs from a variety of organisms were subsequently purified as a consequence of recombinant-DNA approaches (144)(145)(146)(147). UroSs are generally monomeric species with molecular masses of around 30 kDa.…”