Characterization of a low-relative-molecular-mass prolyl 4-hydroxylase from the green alga Chlamydomonas reinhardii

Abstract

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.

The Crystal Structure of an Algal Prolyl 4-Hydroxylase Complexed with a Proline-rich Peptide Reveals a Novel Buried Tripeptide Binding Motif

“…Poly(L-proline) is used as a substrate by Cr-P4H-1 and At-P4H isoenzymes 1 and 2, their K m values for poly(L-proline), M r 5000 -10000, being 140, 2, and 30 M, respectively (11,17,37,38). Poly(Lproline) is not hydroxylated by C-P4Hs, but instead acts as an efficient competitive inhibitor of C-P4H-I with respect to the collagen substrate, the K i being 0.5 M (39).…”

“…Poly(Lproline) is not hydroxylated by C-P4Hs, but instead acts as an efficient competitive inhibitor of C-P4H-I with respect to the collagen substrate, the K i being 0.5 M (39). The (Pro-ProGly) 10 collagen peptide substrate has low affinity for Cr-P4H-1 and At-P4H isoenzyme type 2, the K m values being Ͼ1500 M and 2800 M, respectively, but binds tightly to At-P4H isoenzyme type 1 and C-P4Hs (11,17,37,38). Ser 78 and Ser 87 were mutated to threonine and leucine, respectively, to mimic the C-P4H sequences (Fig.…”

Syncatalytic inactivation of prolyl 4-hydroxylase by anthracyclines

“…(4) An additional argument in favour of a specific action of the anthracyclines comes from the observation that Chlamydomonas reinhardii prolyl 4-hydroxylase was not susceptible to inactivation. Although this enzyme is also an iron-dependent dioxygenase, distinct differences have been found between the algal and vertebrate prolyl 4-hydroxylases (Kaska et al, 1987). Doxorubicin apparently has no affinity for the algal enzyme, as it caused no reversible inhibition.…”

“…In the experiments with Chiamydomonas reinhardii prolyl 4-hydroxylase, the peptide substrate was poly(L-proline), the FeSO4 concentration was 200 /SM, the buffer was Hepes (50 mm, pH 6.8), and the incubation temperature 30°C, these differences being due to the optimal conditions needed by this enzyme (Kaska et al, 1987).…”

“…Highly purified lysyl hydroxllase was prepared by an established method (Turpeenniemi-Hujanen et al, 1980) from a 17-55%-satd.-(NH4)2SO4 fraction of homogenized 14-day chick embryos, by affinity chromatography on concanavalin A-Sepharose and collagen-Sepharose. Prolyl 4-hydroxylase from the green alga Chlamydomonas reinhardii was obtained as described by Kaska et al (1987).…”