Characterisation of the flavin adenine dinucleotide binding region of Myxococcus xanthus protoporphyrinogen oxidase

AbstractProtoporphyrinogen oxidase (PPOX), the penultimate enzyme in the haem biosynthetic pathway catalysers the six electron oxidation of protoporphyrinogen-IX to protoporphyrin-IX, in the presence of flavin adenine dinucleotide (FAD) and oxygen. In humans, partial defects in PPOX result in variegate porphyria. In this study, the FAD binding region in Myxococcus xanthus PPOX was analysed by engineering and characterising a selection of mutant proteins. Amino acid residues which interact with FAD via their side chains were selected for study. Mutants were characterised and compared with wild type protein. Characterisation included FAD quantitation, analysis of FAD spectra and kinetic assay. Results revealed that Serine 20 mutants could still bind FAD, but polarity in this position is favourable, yet not essential for the integrity of FAD binding. Study of Glutamate 39 mutants suggest that a negative charge at position 39 is clearly favoured for interaction with the ribose ring of FAD, as all non-conservative replacements could not bind sufficient FAD. Asparagine 441 appears not to be directly involved in FAD binding but rather in stabilizing the FAD, and polarity in this position appears important. Tryptophan 408 may play a role in orientating or stabilizing the bound substrate during catalysis, and a non-polar (or slightly polar) residue is favoured at this position; however, aromaticity in this position appears not to be critical. Overall this study sheds further light on how M. xanthus PPOX interacts with FAD