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. 2011 Apr 15;6(4):320-4.
doi: 10.1021/cb100298r. Epub 2011 Jan 14.

Tunable, post-translational hydroxylation of collagen Domains in Escherichia coli

Daniel M Pinkas, Sheng Ding, Ronald T Raines, Annelise E Barron

Tunable, post-translational hydroxylation of collagen Domains in Escherichia coli

Daniel M Pinkas et al. ACS Chem Biol. .

Abstract

Prolyl 4-hydroxylases are ascorbate-dependent oxygenases that play key roles in a variety of eukaryotic biological processes including oxygen sensing, siRNA regulation, and collagen folding. They perform their functions by catalyzing the post-translational hydroxylation of specific proline residues on target proteins to form (2S,4R)-4-hydroxyproline. Thus far, the study of these post-translational modifications has been limited by the lack of a prokaryotic recombinant expression system for producing hydroxylated proteins. By introducing a biosynthetic shunt to produce ascorbate-like molecules in Eschericia coli cells that heterologously express human prolyl 4-hydroxylase (P4H), we have created a strain of E. coli that produces collagenous proteins with high levels of (2S,4R)-4-hydroxyproline. Using this new system, we have observed hydroxylation patterns indicative of a processive catalytic mode for P4H that is active even in the absence of ascorbate. Our results provide insights into P4H enzymology and create a foundation for better understanding how post-translational hydroxylation affects proteins.

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Figures

Figure 1
Figure 1. Reactions catalyzed by P4H and ALO
(a) P4H catalyzes the formation of peptidyl 4-hydroxyproline from peptidyl proline and molecular oxygen. (b) In the process of catalysis, the catalytic Fe2+ ion of P4H is oxidized occasionally to Fe3+, which requires reduction by L-ascorbate for catalysis to continue. (c) ALO catalyzes the formation of ascorbate-like molecules from sugar-1,4-lactones. In the case shown, the reaction for which ALO is named, D-arabinono-1,4-lactone is reduced by ALO to D-erythro-ascorbic acid, a five-carbon analog of L-ascorbate.
Figure 2
Figure 2. LC–MS analysis of (Pro-Pro-Gly)5 peptides cytosolically hydroxylated in E. coli under various conditions
(a) UV absorbance chromatograms of (Pro-Pro-Gly)5 peptides from the cultures expressing both P4H and ALO in (“PA1”) Terrific Broth, (“PA2”) M9 minimal medium plus 0.4% w/v tryptone and 0.4% v/v glycerol, and (“PA3”) M9 minimal medium plus 0.4% w/v tryptone. (b) Cultures not expressing ALO: (“NEG”) expressing neither P4H nor ALO in Terrific Broth, (“P1”) expressing P4H only in Terrific Broth, (“P2”) expressing P4H only in M9 minimal medium plus 0.4% w/v tryptone and 0.4% v/v glycerol, and (“P3”) expressing P4H only in M9 minimal medium plus 0.4% w/v tryptone. Arrows indicate number of hydroxylated prolines in the associated peaks as determined by quadrupole mass analysis. Mass spectra of peaks with 0–5 hydroxyls are shown in parts c–h, respectively.
Figure 3
Figure 3. Triple helix formation by P4H-mediated hydroxylation of collagenous peptides in E. coli
(a) Relationship between the Tm values of triple helical (Pro-Pro-Gly)5-foldon and (Pro-Pro-Gly)7-foldon, and their hydroxylation levels. Squares represent (Pro-Pro-Gly)5-foldon. Triangles represent (Pro-Pro-Gly)7-foldon. (b) Hydroxylation levels of (Pro-Pro-Gly)5, (Pro-Pro-Gly)5-foldon, (Pro-Pro-Gly)7, (Pro-Pro-Gly)7-foldon, (Pro-Pro-Gly)10, and (Pro-Pro-Gly)10-foldon constructs co-expressed with both P4H and ALO in E. coli, using M9 minimal medium plus 0.4% w/v tryptone and 0.4% v/v glycerol. Hydroxylation level is reported as the percentage of substrate prolines (proline in the Y position of X-Y-glycine repeats) that were hydroxylated.
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