title: Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation.
abstract: The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolyl-hydroxylation in human hypoxia sensing has ancient origins.
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom;
authors.forename: John S
authors.initials: JS
authors.lastname: Scotti
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom;
authors.forename: Ivanhoe K H
authors.initials: IK
authors.lastname: Leung
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom; Chinese Academy of Medical Sciences, Beijing 100005, China;
authors.forename: Wei
authors.initials: W
authors.lastname: Ge
authors.affiliation: Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom;
authors.forename: Michael A
authors.initials: MA
authors.lastname: Bentley
authors.affiliation: Department of Zoology, University of Oxford, Oxford OX1 3PS, United Kingdom;
authors.forename: Jordi
authors.initials: J
authors.lastname: Paps
authors.affiliation: Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom; and.
authors.forename: Holger B
authors.initials: HB
authors.lastname: Kramer
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom;
authors.forename: Joongoo
authors.initials: J
authors.lastname: Lee
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom;
authors.forename: WeiShen
authors.initials: W
authors.lastname: Aik
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom;
authors.forename: Hwanho
authors.initials: H
authors.lastname: Choi
authors.affiliation: Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom;
authors.forename: Steinar M
authors.initials: SM
authors.lastname: Paulsen
authors.affiliation: Department of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.
authors.forename: Lesley A H
authors.initials: LA
authors.lastname: Bowman
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom;
authors.forename: Nikita D
authors.initials: ND
authors.lastname: Loik
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom; Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom; and.
authors.forename: Shoichiro
authors.initials: S
authors.lastname: Horita
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom;
authors.forename: Chia-hua
authors.initials: CH
authors.lastname: Ho
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom;
authors.forename: Nadia J
authors.initials: NJ
authors.lastname: Kershaw
authors.affiliation: Department of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.
authors.forename: Christoph M
authors.initials: CM
authors.lastname: Tang
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom;
authors.forename: Timothy D W
authors.initials: TD
authors.lastname: Claridge
authors.affiliation: Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom;
authors.forename: Gail M
authors.initials: GM
authors.lastname: Preston
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom;
authors.forename: Michael A
authors.initials: MA
authors.lastname: McDonough
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom; christopher.schofield@chem.ox.ac.uk.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
mesh_terms: D016825:Chlamydomonas reinhardtii / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism*; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000378:metabolism*; D011392:Proline / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016958:Pseudomonas putida / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011392:Proline; C490190:EGLN1 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D010444:Peptide Elongation Factor Tu; D010100:Oxygen