A primitive pathway of porphyrin biosynthesis and enzymology in Desulfovibrio vulgaris

Culture of Desulfovibrio vulgaris in a medium supplemented with 5-aminolevulinic acid and l-methionine-methyl-d(3) resulted in the formation of porphyrins (sirohydrochlorin, coproporphyrin III, and protoporphyrin IX) in which the methyl groups at the C-2 and C-7 positions were deuterated. A previously unknown hexacarboxylic acid was also isolated, and its structure was determined to be 12,18-didecarboxysirohydrochlorin by mass spectrometry and (1)H NMR. These results indicate a primitive pathway of heme biosynthesis in D. vulgaris consisting of the following enzymatic steps: (i) methylation of the C-2 and C-7 positions of uroporphyrinogen III to form precorrin-2 (dihydrosirohydrochlorin); (ii) decarboxylation of acetate groups at the C-12 and C-18 positions of precorrin-2 to form 12,18-didecarboxyprecorrin-2; (iii) elimination of acetate groups of the C-2 and C-7 positions of 12,18-didecarboxyprecorrin-2 to form coproporphyrinogen III; and (iv) conversion of coproporphyrinogen III to protoporphyrin IX via protoporphyrinogen IX. We isolated the following three enzymatic activities involved in steps i–iii from the soluble fraction of the cells by anion-exchange chromatography: S-adenosyl-l-methionine:uroporphyrinogen III methyltransferase, precorrin-2 12,18-acetate decarboxylase, and 12,18-didecarboxyprecorrin-2 2,7-decarboxymethylase; all enzymic products were converted into autooxidized methyl esters and analyzed by thin-layer chromatography, UV–visible (UV-VIS) absorption, and mass spectrometry. The enzymatic reactions in D. vulgaris shed new light on porphyrin biosynthesis at an early stage in the evolution of prokaryotes.