title: Tunable, post-translational hydroxylation of collagen Domains in Escherichia coli.
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.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Pinkas
authors.forename: Sheng
authors.initials: S
authors.lastname: Ding
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Raines
authors.forename: Annelise E
authors.initials: AE
authors.lastname: Barron
mesh_terms: D001205:Ascorbic Acid / Q000235:genetics / Q000378:metabolism*; D002853:Chromatography, Liquid; D003094:Collagen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D006900:Hydroxylation; D006909:Hydroxyproline / Q000378:metabolism; D013058:Mass Spectrometry; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D014169:Transformation, Bacterial
chemical_list: D003094:Collagen; D011392:Proline; D011393:Procollagen-Proline Dioxygenase; D001205:Ascorbic Acid; D006909:Hydroxyproline