Abstract
4-Hydroxyproline is found in collagens, collagen-like proteins, elastin, and the hypoxia-inducible transcription factor in animals and in many hydroxyproline-rich glycoproteins in plants. We report here on the cloning and characterization of a second plant P4H (prolyl 4-hydroxylase), At-P4H-2, from Arabidopsis thaliana. It consists of 299 amino acids and shows 33% sequence identity to the first characterized isoenzyme, At-P4H-1. A characteristic feature of the At-P4H-2 polypeptide is a 49-amino-acid C-terminal toxin homology domain with 6 cysteines that is not found in At-P4H-1 but is present in a putative rice P4H homologue. At-P4H-2 differed distinctly from At-P4H-1 in its substrate specificity. Recombinant At-P4H-2 hydroxylated poly(L-proline) and extensin and arabinogalactan-like peptides effectively but with much higher Km values than At-P4H-1, suggesting different roles for the two At-P4Hs in the plant cell. Unlike At-P4H-1, At-P4H-2 hydroxylated collagen-like peptides only very inefficiently and did not hydroxylate hypoxia-inducible transcription factor alpha-like peptides at all. All the peptides efficiently hydroxylated by At-P4H-2 had at least 3 consecutive prolines, suggesting that these may represent a minimum requirement for efficient hydroxylation by this isoenzyme. N-terminal sequencing of an extensin-like peptide SPPPVYKSPPPPVKHYSPPPV indicated that At-P4H-2 preferentially hydroxylated the 3rd proline in the C-terminal PPP triplet. The Km values of At-P4H-2 for the reaction cosubstrates Fe2+, 2-oxoglutarate, and ascorbate were similar to those of At-P4H-1 with the exception that the Km for iron was about 3-fold lower. Pyridine-2,4-dicarboxylate and pyridine-2,5-dicarboxylate, well known competitive inhibitors of the vertebrate P4Hs with respect to 2-oxoglutarate, were also competitive inhibitors of At-P4H-2 but with Ki values 5-100-fold higher than those of human type I collagen P4H. It thus seems that there are some distinct differences in the structure of the 2-oxoglutarate-binding site between At-P4H-2 and the animal collagen P4Hs.
Highlights
4-Hydroxyproline is found in animal proteins almost exclusively in collagens, elastin, and more than 20 additional pro
We have cloned a second 299-amino-acid A. thaliana P4H based on the GenBankTM sequence NP566279.1 and have named it At-P4H-2, its gene being located in chromosome 3
This polypeptide corresponds to AAF08583 as identified in the first sequence homology search (15), the first 178 amino acids encoded by NP566279.1 being identical to those of AAF08583, whereas the subsequent AAF08583 sequence has a deletion corresponding to 21 amino acids
Summary
4-Hydroxyproline is found in animal proteins almost exclusively in collagens, elastin, and more than 20 additional pro-. The function of 4-hydroxyproline residues in collagens is to stabilize their triplehelical structure at body temperature (1, 2) Another family of cytoplasmic and nuclear P4Hs that act on the ␣ subunit of the hypoxia-inducible transcription factor (HIF) has been cloned and characterized from the vertebrates and Caenorhabditis elegans and Drosophila melanogaster (8 – 10). These HIF-P4Hs play a key role in the regulation of oxygen homeostasis by hydroxylating single proline residues in LXXLAP sequences, the 4-hydroxyproline residue formed acting as a signal for the proteasomal degradation of HIF␣ (8 –10). The recombinant enzyme is a 29kDa monomer that efficiently hydroxylates poly(L-proline) and many synthetic peptides corresponding to proline-rich repeats in plant glycoproteins and other proteins (15) It effectively hydroxylated collagen-like and HIF␣-like peptides (15). A monomeric P4H has been cloned from the Paramecium bursaria Chlorella virus-1 (PBCV-1) and has like-
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