The Active Site of an Algal Prolyl 4-Hydroxylase Has a Large Structural Plasticity

Abstract

Prolyl 4-hydroxylases (P4Hs) are 2-oxoglutarate dioxygenases that catalyze the hydroxylation of peptidyl prolines. They play an important role in collagen synthesis, oxygen homeostasis, and plant cell wall formation. We describe four structures of a P4H from the green alga Chlamydomonas reinhardtii, two of the apoenzyme at 1.93 and 2.90 Å resolution, one complexed with the competitive inhibitor Zn 2؉ , and one with Zn 2؉ and pyridine 2,4-dicarboxylate (which is an analogue of 2-oxoglutarate) at 1.85 Å resolution. The structures reveal the double-stranded ␤-helix core fold (jellyroll motif), typical for 2-oxoglutarate dioxygenases. The catalytic site is at the center of an extended shallow groove lined by two flexible loops. Mutagenesis studies together with the crystallographic data indicate that this groove participates in the binding of the proline-rich peptide-substrates. It is discussed that the algal P4H and the catalytic domain of collagen P4Hs have notable structural similarities, suggesting that these enzymes form a separate structural subgroup of P4Hs different from the hypoxia-inducible factor P4Hs. Key structural differences between these two subgroups are described. These studies provide first insight into the structure-function relationships of the collagen P4Hs, which unlike the hypoxiainducible factor P4Hs use proline-rich peptides as their substrates.

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

“…In contrast, the plant P4Hs and probably also the HIF-P4Hs are monomers (2,10). Plant P4Hs have a ϳ30% sequence identity to the catalytic domain of the C-P4H ␣-subunits (11,12), and they also resemble C-P4Hs in that they hydroxylate proline-rich polypeptides and are located in the lumen of the endoplasmic reticulum (2). HIF-P4Hs, on the other hand, are cytoplasmic and nuclear enzymes (8,9) that act on proline residues in -Leu-X-X-Leu-Ala-Pro-motifs in HIF-␣ (13,14).…”

“…The catalytic sites of all P4Hs have the conserved -His-X-Asp-…-His-catalytic motif for Fe 2ϩ coordination and a basic residue that binds the C5 carboxylate moiety of 2OG. The crystal structures of two P4Hs have been solved, namely those of human HIF-P4H-2 (10) and a plant P4H from Chlamydomonas reinhardtii (Cr-P4H-1) (12). The structures of these two enzymes share the jelly-roll core fold preceded by an N-terminal part that contains two long ␣-helices in both structures, and also three ␤-strands in Cr-P4H-1 and 2 ␤-strands in HIF-P4H-2 (10,12).…”

Missense Mutations That Cause Bruck Syndrome Affect Enzymatic Activity, Folding, and Oligomerization of Lysyl Hydroxylase 2

“…Mutation of the corresponding serine in recombinant human LH1 has been shown to reduce its activity by ϳ30%, although it does not affect the K m for 2-oxoglutarate (8). Direct hydrogen bonding between the C-5 carboxylate of 2-oxoglutarate or its analog and the side chains of a threonine in the fourth strand of the jellyroll fold and a tyrosine in the first strand is also common in the enzyme superfamily (31,34). As the three-dimensional structure of LH is as yet unknown, the exact structural role of Thr 604 in 2-oxoglutarate binding remains to be established.…”

“…This basic residue is located in position ϩ9 or ϩ10 with respect to the second Fe 2ϩ -binding histidine in all 2-oxoglutarate dioxygenases that have been characterized so far except for the asparaginyl hydroxylase that hydroxylates the hypoxia-inducible factor, where it is located between the Fe 2ϩ -binding aspartate and the second histidine in the fourth strand (32). In many 2-oxoglutarate dioxygenases, additional interactions with the C-5 carboxylate have been shown to be provided by a side chain of a serine or threonine located in position ϩ2 or ϩ4, respectively, relative to the basic residue in the eighth strand (31,34). Mutation of the corresponding serine in recombinant human LH1 has been shown to reduce its activity by ϳ30%, although it does not affect the K m for 2-oxoglutarate (8).…”

“…The mutations identified in Bruck syndrome are all close to each other but at some distance from His 662 , Asp 664 , and His 714 , which correspond to the three conserved Fe 2ϩ -binding residues identified in LH1 and several other 2-oxoglutaratedependent dioxygenases, and Arg 724 , which corresponds to the conserved basic residue required for binding the C-5 carboxyl group of 2-oxoglutarate (see Fig. 1) (7,8,(31)(32)(33)(34). To study the functional and structural consequences of the R594H, G597V, and T604I LH2(long) mutations found in Bruck syndrome 2 patients at the molecular level, we expressed wild-type and mutant recombinant LH2(long) polypeptides in insect cells, purified them to homogeneity, and analyzed their folding and catalytic properties.…”