| Protoporphyrinogenâ…¨oxidase (PPO, EC 1.3.3.4) converts protoporphyrinogenâ…¨to protoporphyrinâ…¨, playing an important part in the heme/chlorophyll biosynthetic pathway. In human, the partial deficiency of PPO causes an inherited human disease called variegated prophyria (VP). In plants, the inhibition of PPO leads to cell death in the presence of light. This special characteristic of PPO enzymes makes it to be an important target both in agriculture and medicine.Bacillus subtilis PPO (bsPPO) is unique among this protein family. The soluble monomer nature, the lack of significant inhibition by acifluorfen and the ability to utilize protoporphyrinogenâ…¨, mesoporphyrinogenâ…¨, and coproporphyrinogenâ…¢set bsPPO in a unique class, but make it an ideal candidate for mechanistic studies.Here we present the crystal structure of bsPPO bound to AF at 2.9 A resolution. Our structure shows that the AF molecule binds to a new site outside the previously identified inhibitor binding pocket. Most importantly, the benzene ring of the 2-nitrobenzoic acid moiety of AF lies parallel to the isoalloxazine ring of FAD at a distance of less than 3.5 A, providing a framework for the interaction of FAD with the substrate protoporphyrinogenâ…¨. Furthermore, our structure reveals that the larger substrate binding chamber and predominantly positively charged chamber surface of bsPPO are more favorable for the binding of coproporphyrinogenâ…¢. Finaly, on the basis of crystal structure, we modeled the binding of the substrate protoporphyrinogenâ…¨, and analyzed the activities of some mutations around active site. The experimental results were quite consistent with our structure prediction.In summary, our crystal structure of bsPPO bound to AF explains its biochemically unique properties, provides structural evidence for the catalytic mechanism proposed by Koch et al, and increases several important informations for understanding of this vital protein family.
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