PARTIAL CHARACTERISATION OF RECOMBINANT MYXOCOCCUS XANTHUS AND HUMAN PROTOPORPHYRINOGEN OXIDASES, AND THEIR PHYLOGENETIC ANALYSES USING PROTEIN SEQUENCES FROM DIVERSE SPECIES

Siziba KB1, Corrigall AV1, Gamieldien J2, Maneli MH1, Kirsch RE3, Meissner PN1

 

1Lennox Eales Porphyria Laboratories, Liver Centre, University of Cape Town, 2South African National Bioinformatics Institute, University of the Western Cape, 3Dept. of Medicine, Liver Centre, University of Cape Town, South Africa

 

 

A kinetic and inhibitor characterisation of PPOX from the prokaryote Bacillus subtilis has previously been reported from our laboratory. Here, in an extended study, a partial characterisation of PPOX from another prokaryote Myxococcus xanthus, in addition to the human form, is reported. The required recombinant PPOXs were expressed in E. coli cells and purified to apparent homogeneity by rapid metal chelate affinity chromatography. Thereafter, detailed kinetic and inhibitor profiles were established for each form of the enzyme. Sequence data (nucleotide and/or protein/polypeptide) accessed from publicly available databases allowed us to perform multiple protein sequence alignment and phylogenetic analyses in order to further analyse the possible relationships and/or differences between the biochemical properties amongst these PPOXs and those from several diverse species. Detailed kinetic analysis showed that M. xanthus PPOX behaved more like human PPOX than the B. subtilis enzyme - M. xanthus had a more stringent substrate specificity compared to that of the B. subtilis enzyme and, secondly, both M. xanthus and human PPOXs were were very sensitive to diphenylether inhibition, unlike B. subtilis. Our data suggests that differences in inhibitor profiles are not simply general differences between prokaryotic and eukaryotic forms of PPOX, nor does it simply reflect the Gram positive/negative status of the organism. Sequence alignment analyses identified, in most cases, two conserved domains (A and B), both of which were not present in the human monoamine oxidase (MAO-B) sequence, used to root the PPOX proteins for phylogenetic analysis. From this work it is clear that the N-terminal domain (A) appears to be better conserved in most of the sequences, than the C-terminal domain (B). These differences shed some light on their behaviour, particularly with respect to the different inhibitors - PPOXs containing both the conserved domains (A and B), appear to have greater affinity for the diphenylether inhibitors and tighter substrate specificity. The phylogenetic tree showed interesting relationships and clustering. For example, all plant PPOXs were clustered together, but two distinct branches were formed, which separated these proteins on the basis of the organelle in which the PPOX isoform is located (mitochondria or chloroplast). Amongst the prokaryotic PPOXs there was an important deviation from the bacterial clustering. M. xanthus PPOX was more closely associated with the eukaryotic (yeast and mammalian) proteins than other prokaryotic PPOXs. Our biochemical findings add weight to this anomaly in that the kinetic behaviour of this enzyme was similar to that of eukaryotic enzymes. In conclusion, we provide evidence that differences in the biochemical properties of various PPOXs, although sometimes relatively subtle, probably relate to their differences at the primary structural level which have evolved over time. PPOXs containing both conserved sequence homologies (domains A and B) appear to have greater affinity and catalytic activity towards the substrate, protoporphyrinogen-IX and are also more sensitive to the diphenylether inhibitors