Characterization of a second Arabidopsis thaliana prolyl 4-hydroxylase with distinct substrate specificity.
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.
Tiainen
Päivi
P
Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, University of Oulu, FIN-90014 Oulu, Finland.
Myllyharju
Johanna
J
Koivunen
Peppi
P
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology*; D002460:Cell Line; D003001:Cloning, Molecular; D003094:Collagen / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005685:Galactans / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D006900:Hydroxylation; D006909:Hydroxyproline / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007656:Ketoglutaric Acids / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000031:analogs & derivatives / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D018411:Spodoptera; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism
D004791:Enzyme Inhibitors; D005685:Galactans; D006023:Glycoproteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007656:Ketoglutaric Acids; D010455:Peptides; D010940:Plant Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C005488:extensin protein, plant; D003094:Collagen; D011392:Proline; D011393:Procollagen-Proline Dioxygenase; D006909:Hydroxyproline; C005653:arabinogalactan