Biosynthesis of porphyrins and related macrocycles. Part 41. Fate of oxygen atoms as precorrin-2 carrying eight labelled carboxyl groups (13C18O2H) is enzymatically converted into cobyrinic acid

Abstract

5-Amino[1,4-¹³C₂]laevulinic acid and 5-amino[1-¹³C]laevulinic acid are synthesised and all three ¹⁶O atoms of the latter are exchanged for ¹⁸O. The ¹³C,¹⁸O-labelled material is then converted in vitro into precorrin-2 2 by the combined action of four genetically overproduced enzymes. The product is isolated in its aromatised form, sirohydrochlorin 10 and ¹³C-NMR shows that all 8 carboxyl groups of 10 retain both oxygen atoms throughout the biosynthesis. A cell-free enzyme preparation from Propionibacterium shermanii converts the ¹³C,¹⁸O-labelled sirohydrochlorin 10via2 into cobyrinic acid 4, a late precursor of vitamin B₁₂. ¹³C-NMR proves that 6 carboxyl groups of cobyrinic acid (b–g, inclusive) retain both oxygen atoms whereas the a-carboxyl group undergoes specific loss of one labelled oxygen atom.