Characterization of a low-relative-molecular-mass prolyl 4-hydroxylase from the green alga Chlamydomonas reinhardii.
Prolyl 4-hydroxylase was partially purified and characterized from the unicellular green alga, Chlamydomonas reinhardii. This enzyme differed from all the animal and plant prolyl 4-hydroxylases studied so far in that its Mr was only about 40,000 by gel filtration, being thus less than one-sixth of those determined for the vertebrate and higher-plant enzymes. The algal enzyme did not hydroxylate to any significant extent chick-embryo protocollagen or triple-helical (Pro-Pro-Gly)10, whereas a low hydroxylation rate was found with denatured (Pro-Pro-Gly)10. Poly(L-proline), which is an effective inhibitor of the vertebrate enzymes but acts as a substrate for some higher-plant enzymes, was a good substrate. In the absence of poly(L-proline) the enzyme catalysed an uncoupled decarboxylation of 2-oxoglutarate. Studies of the Km values for the co-substrates and cofactors and the specificity of the 2-oxoglutarate requirement, as well as inhibition studies with selected 2-oxoglutarate analogues, suggested that the catalytic site of the algal enzyme is similar to, but not identical with, those of the vertebrate enzymes. The existence of distinct similarities was further demonstrated by an inhibition of the algal enzyme activity with a monoclonal antibody to the beta-subunit of human prolyl 4-hydroxylase. The amount of prolyl 4-hydroxylase activity in the algal cells was not altered by signals which recognize the presence or absence of the cell wall, as determined in studies on experimental cell-wall regeneration and wall-less mutants.
D002473:Cell Wall / Q000378:metabolism; D002696:Chlamydomonas / Q000201:enzymology*; D002853:Chromatography, Liquid; D003429:Cross Reactions; D006909:Hydroxyproline / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D013379:Substrate Specificity
D007656:Ketoglutaric Acids; D010455:Peptides; D011393:Procollagen-Proline Dioxygenase; D006909:Hydroxyproline