the result that the methyl group that had been added to ring C is isomerised to its final location, an example of intramolecular transfer: precorrin-8X hydrogenobyrinate From hydrogenobyrinic acid to hydrogenobyrinic acid a,c-diamide The next enzyme in the pathway, CobB, selectively converts two of the eight carboxylic acid groups into their primary amides. ATP is used to provide the energy for amide bond formation, with the transferred ammonia coming from glutamine: hydrogenobyrinic acid + 2 ATP + 2 glutamine + 2 H2O hydrogenobyrinic acid a,c-diamide + 2 ADP + 2 phosphate + 2 glutamic acid From hydrogenobyrinic acid a,c-diamide to acid a,c-diamide Cobalt(II) insertion into the macrocycle is catalysed by the enzyme Cobalt chelatase (CobNST): hydrogenobyrinic acid a,c-diamide + Co2+ + ATP + H2O acid a,c-diamide + ADP + phosphate + H+ It is at this stage that the aerobic pathway and the anaerobic pathway merge, with later steps being chemically identical. Detail of steps from uroporphyrinogen III to a,c-diamide in anaerobic organisms Many of the steps beyond uroporphyrinogen III in anaerobic organisms such as Bacillus megaterium involve chemically similar but genetically distinct transformations to those in the aerobic pathway. From precorrin-2 to cobalt-sirohydrochlorin The key difference in the pathways is that cobalt is inserted early in anaerobic organisms by first oxidising precorrin-2 to its fully aromatised form sirohydrochlorin and then to that compound's cobalt(II) complex. These reactions are catalysed by CysG and Sirohydrochlorin cobaltochelatase. From cobalt-sirohydrochlorin to cobalt-factor III As in the aerobic pathway, the third methyl group is introduced by a methyltransferase enzyme, CbiL: cobalt-sirohydrochlorin + S-adenosyl methionine cobalt-factor III + S-adenosyl-L-homocysteine From cobalt-factor III to cobalt-precorrin-4 Methylation and ring contraction to form the corrin macrocycle occurs next, catalysed by the enzyme Cobalt-factor III methyltransferase (CbiH, ) cobalt-factor III + S-adenosyl methionine cobalt-precorrin-4 + S-adenosyl-L-homocysteine In this pathway, the resulting material contains a δ-lactone, a six-membered ring, rather than the γ-lactone (five-membered ring) of precorrin-3B. From cobalt-precorrin-4 to cobalt-precorrin-5A The introduction of the methyl group at C-11 in the next step is catalysed by Cobalt-precorrin-4 methyltransferase (CbiF, ) cobalt-precorrin-4 + S-adenosyl methionine cobalt-precorrin-5 + S-adenosyl-L-homocysteine From cobalt-precorrin-5A to cobalt-precorrin-5B The scene is now set for the extrusion of the two-carbon fragment corresponding to the acetate released in the formation of precorrin-6A in the aerobic pathway. In this case the fragment released is acetaldehyde and this is catalysed by CbiG: cobalt-precorrin-5A + H2O cobalt-precorrin-5B + acetaldehyde + 2 H+