After the formation of anhydrotetracycline, ATC monooxygenase ("OxyS") oxidizes the C-6 position in an enantioselective manner in the presence of the cofactor NADPH and atmospheric oxygen to produce 5a,11a-dehydrotetracycline. Next, a hydroxylation occurs at the C-5 position of 5a,11a-dehydrotetracycline via the oxygenase encoded as "OxyE" in the oxytetracycline gene cluster. This produces the intermediate 5a,11a-dehydro-oxytetracycline. However, the exact mechanism of this step remains to be unclear. The final step of this biosynthesis occurs through the reduction of a double bond in the α, β - unsaturated ketone of 5a,11a-dehydro-oxytetracycline. In this final step, the cofactor NADPH is employed by "TchA" (reductase) as the reducing agent. Upon reduction, the enol form is favored due to conjugation, thus producing the aromatic polyketide oxytetracycline. Figure 2 shows the biosynthesis as described above, as well as an arrow-pushing mechanism of NADPH being used as the final cofactor in the biosynthesis of oxytetracycline.