High-performance liquid chromatography of coproporphyrin isomers.

A reversed-phase system is described for the simultaneous isocratic separation of coproporphyrin I, II, III and IV isomers. The retention behaviour of coproporphyrin I and III is studied in detail. The method is suitable for both analytical and semi-preparative separation.     

Production of copper coproporphyrin III by Bacillus cereus. I. Purification and identification of copper coproporphyrin III.

Bacillus cereus strains 2 and T did not form spores and accumulated a large amount of purple pigment inside the cells, when cultured in a yeast extract-ammonium salt medium with excess glucose. The pigment was extracted and crystallized as the ethyl ester. It was identified as copper coproporphyrin III.     

Production of copper coproporphyrin III by Bacillus cereus. II. Regulation of the biosynthesis of coproporphyrin III and its copper complex by oxygen and heavy metal ions.

The effects of oxygen and heavy metal ions on the production of copper coproporphyrin III were studied in Bacillus cereus strain 2. The formation of copper coproporphyrin III was found to be maximum when the cells were cultivated in G-medium at a low level of oxygen supply, but it was suppressed at extremely low oxygen supply levels. When the cells were cultured in metal-free G-medium, neither metal-coproporphyrin III nor coproporphyrin III was formed. In the presence of copper in the medium (400-100 micrometers), the formation of coproporphyrin III copper salt was maximum, but the addition of various heavy metal ions other than copper to the copper-free medium resulted in the formation of neither coproporphyrin III nor its metal chelates. Copper ions appear to be specifically required for coproporphyrin III formation.     

Uroporphyrin- and coproporphyrin I-accumulating mutant of Escherichia coli K12.

A new type of haem-deficient mutant was isolated in Escherichia coli K12 by neomycin selection. The mutant was deficient in uroporphyrinogen III cosynthase activity as indicated by the accumulation of uroporphyrin I and coproporphyrin. The mapping of the corresponding hemD gene by P1-mediated transduction showed that the new gene was located between ilv and cya, at min 83 on the chromosomal map of Escherichia coli K12.     

A mutant of Escherichia coli which accumulates large amounts of coproporphyrin.

A mutant of Escherichia coli which accumulates a large amount of coproporphyrin, presumably because of a block in heme biosynthesis, has been isolated after nitrosoguanidine mutagenesis. On rich media, the mutant forms colonies which give bright orange fluorescence when illuminated with ultraviolet light. The mutant appears to be similar to a Salmonella typhimurium mutant, deficient in uroporphyrinogen III cosynthase, described by Sasarman and Desrochers ((1976) J. Bacteriol. 128, 717--721). A striking property of the mutant is that coproporphyrin is retained within the cells in rich media but is almost totally excreted out of cells in minimal glucose medium.     

Active site architecture of coproporphyrin ferrochelatase with its physiological substrate coproporphyrin III: Propionate interactions and porphyrin core deformation

Coproporphyrin ferrochelatases (CpfCs) are enzymes catalyzing the penultimate step in the coproporphyrin-dependent (CPD) heme biosynthesis pathway, which is mainly utilized by monoderm bacteria. Ferrochelatases insert ferrous iron into a porphyrin macrocycle and have been studied for many decades, nevertheless many mechanistic questions remain unanswered to date. Especially CpfCs, which are found in the CPD pathway, are currently in the spotlight of research. This pathway was identified in 2015 and revealed that the correct substrate for these ferrochelatases is coproporphyrin III (cpIII) instead of protoporphyrin IX, as believed prior the discovery of the CPD pathway. The chemistry of cpIII, which has four propionates, differs significantly from protoporphyrin IX, which features two propionate and two vinyl groups. These findings let us to thoroughly describe the physiological cpIII-ferrochelatase complex in solution and in the crystal phase. Here, we present the first crystallographic structure of the CpfC from the representative monoderm pathogen Listeria monocytogenes bound to its physiological substrate, cpIII, together with the in-solution data obtained by resonance Raman and UV–vis spectroscopy, for wild-type ferrochelatase and variants, analyzing propionate interactions. The results allow us to evaluate the porphyrin distortion and provide an in-depth characterization of the catalytically-relevant binding mode of cpIII prior to iron insertion. Our findings are discussed in the light of the observed structural restraints and necessities for this porphyrin-enzyme complex to catalyze the iron insertion process. Knowledge about this initial situation is essential for understanding the preconditions for iron insertion in CpfCs and builds the basis for future studies.     

The process of continuous production and isolation of coproporphyrin

Summary The production of coproporphyrin from glucose and urea has been achieved by semi-aerobic culture ofSaccharomyces cerevisiae. The cells were entrapped in alginate gel and packed into a column reactor for continuous long-term process. The porphyrins were isolated and simultaneously concentrated from the effluent by liquid-solid phase extraction, purified by liquid chromatography and finally crystallized. Using the process described the final product of high purity was obtained.