Procollagen prolyl-4-hydroxylase (P4H) catalyzes the conversion of peptidyl proline to trans-4-hydroxyproline (Hyp). P4H is a member of the α-ketoglutarate-dependent mononuclear non-heme iron oxygenase (αKG/Fe(II)-oxygenase) family of enzymes, which catalyze a wide variety of reactions. The genome of Bacillus anthracis (B. anthracis), the causative agent of anthrax, contains a gene that is annotated as a p4h based on the predicted amino acid sequence. Recently the immunodominant protein of the B. anthracis exosporium has been identified as BclA. BclA has collagen-like repeat sequences and has a triple helical structure similar to that of animal collagens. We have detected Hyp from the protein extracts of spores of B. anthracis and this is the first report for Hyp detection in bacteria. These results strongly support that B. anthracis contains a P4H-like protein, anthrax-P4H.

We have expressed and purified a recombinant form of the putative anthrax-P4H and found that it is a homodimer of 24.3 kDa subunit. The anthrax-P4H exhibits activity and UV-vis spectroscopic properties characteristic of an αKG/Fe(II)-oxygenase, and it can bind a collagen-like substrate, (Gly-Pro-Pro)₁₀, with an affinity comparable to that of human type (I) collagen-P4H (human-P4H-1). This is the first report of any P4H-like protein from a bacterial source. We propose that anthrax-P4H can serve as a model for human-P4H-1 from its substrate specificity and kinetic parameters comparable to human-P4H-1.

We also report the crystal structure of anthrax-P4H to 1.40 Å. The structure reveals the double stranded β-helix core fold (jellyroll) motif, characteristic of Fe(II)/αKG oxygenases. The oligomerization of the protein is a homodimeric, α2, structure, which makes the anthrax-P4H a new member to the P4H family of enzymes. A putative peptide-binding groove has been proposed based on that identified in the structure of P4H from Chlamydomonas reinhardtii (green alga) (Cr-P4H-1). The anthrax-P4H structure provides insight into the dimeric structure of the α-subunits of human-P4H-1, as well as to help understand the mode of substrate recognition that may aide in the development of selective inhibitors.