Crystal Structure of Prolyl 4-Hydroxylase from Bacillus anthracis

Abstract

Prolyl 4-hydroxylases (P4H) catalyze the posttranslational hydroxylation of proline residues and play a role in collagen production, hypoxia response, and cell wall development. P4Hs belong to the Fe (II)/αKG oxygenases and require Fe(II), α-ketoglutarate (αKG), and O 2 for activity. We report the 1.40 Å structure of a P4H from Bacillus anthracis, the causative agent of anthrax, whose immunodominant exosporium protein BclA contains collagen-like repeat sequences. The structure reveals the double stranded β-helix core fold characteristic of Fe(II)/αKG oxygenases. This fold positions Fe-binding and αKG-binding residues in what is expected to be catalytically-competent orientations and is consistent with proline peptide substrate binding at the active site mouth. Comparisons of the anthrax-P4H structure with Cr-P4H-1 structures reveal similarities in a peptide surface groove. However, sequence and structural comparisons suggest differences in conformation of adjacent loops may change the interaction with peptide substrates. These differences may be the basis of substantial disparity between the K M values for the Cr-P4H-1 vs. the anthrax and human P4H enzymes. Additionally, while previous structures of P4H enzymes are monomers, Bacillus anthracis P4H forms an α 2 homodimer and suggests residues important for interactions between the α 2 subunits of the α 2 β 2 human collagen P4H. Thus the anthrax-P4H structure provides insight into the structure and function of the α subunit of human-P4H, which may aid in the development of selective inhibitors of the human-P4H enzyme involved in fibrotic disease.Prolyl 4-hydroxylase (P4H) enzymes are involved in the post-translational formation of trans-4-hydroxyproline (Hyp) from peptidyl proline. In plants, P4H enzymes act on prolines present in extensins, in proline-rich proteins, and in arabinogalactan proteins to form hydroxyproline-rich glycoproteins (HRGPs) that stabilize plant cell walls (1,2). Vertebrates † This work was supported, in whole or in part, by National Institutes of Health Grants GM079446 (JL), 5P20 RR17708 (COBRE Center in Protein Structure and Function) (JL), and T2 GM 08454 (MAC).*Address correspondence to: Emily Scott, 1251 Wescoe Hall Dr., Lawrence, KS 66045. Tel.: 785-864-5559; Fax: 785-864-5326; eescott@ku.edu. ‡ Deceased, August 14, 2008. § This paper is dedicated in memory of Dr. Julian Limburg. This publication would not have been possible without his unlimited dedication to his students as well as his intellect and passion for science. Those of who studied under his tutelage will carry his cherished memory with us.The atomic coordinates and structure factors (code 3ITQ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). SUPPORTING INFORMATIONAn alignment of the anthrax, human, and algal prolyl 4-hydroxylase amino acid sequences is provided as supplemental material. This alignment is annotated with conserved amino acids, secondary...

Characterization of BcaA, a Putative Classical Autotransporter Protein in Burkholderia pseudomallei

“…Prolyl 4-hydroxylase, which catalyzes the most prevalent posttranslational modification in humans, has as the substrate collagen, functioning by stabilizing the collagen triple-helix structure (40), elastin, where it affects the formation of elastin fibrils (41), and even prion proteins, although the consequences of the presence of the prion proteins are unknown (39). In bacteria, a Bacillus anthracis enzyme designated anthrax-P4H has been shown to hydroxylate peptidyl prolines; however, although it binds to collagen-like peptides in vitro, its physiological substrate and role remain unknown (42). Further studies should include determining if bcaB plays a role in hydroxylating any of the 52 prolines found in BcaA, since it is possible that BcaA is the physiological substrate for BcaB, which would be consistent with their operon structures.…”

Bacillus anthracis Prolyl 4-Hydroxylase Modifies Collagen-like Substrates in Asymmetric Patterns

“…The structure illustrates various enzyme-substrate interactions in the binding groove and in the substrate binding loop (SBL) region adjacent to the active site, with the peptide adopting the PPII helix conformation (34,35). These highly mobile SBL regions are conserved through P4Hs and in the absence of a substrate one of the loops is usually disordered (30,36,37).…”

“…On the other hand, Bacillus anthracis P4H (BaP4H) is unique among bacterial P4Hs in that it is reported to bind the human collagen peptide (P-P-G) 10 with an affinity similar to that of human C-P4H (1,36,39). A sequence alignment of BaP4H with human C-P4H shows ϳ30% sequence identity.…”

“…A genome-based study of collagen structural motifs in bacterial and viral proteins showed that bacterial collagen structural motifs favor a (G-P-T) triplet unit rather than the second most prevalent (G-P-Q) repeat unit (40). Because the proteome of B. anthracis contains (G-P-T) repeats in some proteins, it has been proposed that a possible physiological substrate for BaP4H could be a protein consisting of (G-P-T) n repeats (36,41). However, the substrate for BaP4H…”

“…The previously reported crystal structure of BaP4H is an apoform of the enzyme lacking necessary cofactors (36,41). To gain insight into the site-specific prolyl hydroxylation activity of BaP4H, it is important to understand how the active site resi-FIGURE 1.…”