Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation John S. Scottia, Ivanhoe K. H. Leunga,1,2, Wei Gea,b,1, Michael A. Bentleyc, Jordi Papsd, Holger B. Kramere, Joongoo Leea, WeiShen Aika, Hwanho Choia, Steinar M. Paulsenc,3, Lesley A. H. Bowmanf, Nikita D. Loika,4, Shoichiro Horitaa,e, Chia-hua Hoa,5, Nadia J. Kershawa,6, Christoph M. Tangf, Timothy D. W. Claridgea, Gail M. Prestonc, Michael A. McDonougha, and Christopher J. Schofielda,7 aChemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom; bChinese Academy of Medical Sciences, Beijing 100005, China; cDepartment of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom; dDepartment of Zoology, University of Oxford, Oxford OX1 3PS, United Kingdom; eDepartment of Physiology, Anatomy, and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom; and fDepartment of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom Edited by Gregg L. Semenza, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved August 5, 2014 (received for review May 30, 2014) The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases Results in eukaryotes include collagen stabilization, hypoxia sensing, and Pseudomonas spp. Contain a Functional PHD. To investigate the role translational regulation. The hypoxia-inducible factor (HIF) sensing of a putative PHD homolog in Pseudomonas aeruginosa (PPHD), system is conserved in animals, but not in other organisms. How- we initially characterized a PPHD insertional mutant strain. ever, bioinformatics imply that 2OG-dependent prolyl-hydroxy- Metabolic screening studies revealed that the PPHD mutant strain lases (PHDs) homologous to those acting as sensing components displays impaired growth in the presence of iron chelators (e.g., for the HIF system in animals occur in prokaryotes. We report 2,2-bipyridine) and produces increased levels of the bacterial vir- cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein ulence factor pyocyanin (SI Appendix,Fig.S1)(12),implying (PPHD) contain a 2OG oxygenase related in structure and function PPHD is involved in iron regulation. Ferrous iron is essential for to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout PHD catalysis and treatment of human cells with iron chelators, mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We Significance identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal The Fe(II)- and 2-oxoglutarate (2OG)-dependent hypoxia- structure of PPHD reveals striking similarity to human PHD2 and inducible transcription factor prolyl-hydroxylases play a central a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal struc- role in human oxygen sensing and are related to other prolyl- ture of PPHD complexed with intact EF-Tu reveals that major con- hydroxylases involved in eukaryotic collagen biosynthesis and formational changes occur in both PPHD and EF-Tu, including ribosomal modification. The finding that a PHD-related prolyl-hy- > a 20-Å movement of the EF-Tu switch I loop. Comparison of droxylase in Pseudomonas spp. regulates pyocyanin biosynthesis the PPHD structures with those of HIF and collagen PHDs reveals supports prokaryotic origins for the eukaryotic prolyl-hydrox- conservation in substrate recognition despite diverse biological ylases. The identification of the switch I loop of elongation factor roles and origins. The observed changes will be useful in designing Tu (EF-Tu) as a Pseudomonas prolyl-hydroxylase domain contain- new types of 2OG oxygenase inhibitors based on various confor- ing protein (PPHD) substrate provides evidence of roles for 2OG mational states, rather than active site iron chelators, which make oxygenases in both translational and transcriptional regulation. A up most reported 2OG oxygenase inhibitors. Structurally in- structure of the PPHD:EF-Tu complex, the first to the authors’ BIOCHEMISTRY formed phylogenetic analyses suggest that the role of prolyl- knowledge of a 2OG oxygenase with its intact protein substrate, hydroxylation in human hypoxia sensing has ancient origins. reveals that major conformational changes occur in both PPHD and EF-Tu and will be useful in the design of new prolyl- he hypoxia-inducible transcription factor (HIF) is a major hydroxylase inhibitors. Tregulator of the response to limited oxygen availability in humans and other animals (1–3). A hypoxia-sensing component of Author contributions: J.S.S., I.K.H.L., W.G., G.M.P., M.A.M., and C.J.S. designed research; J.S.S., I.K.H.L., W.G., M.A.B., J.P., H.B.K., J.L., W.A., H.C., S.M.P., L.A.H.B., N.D.L., C.-h.H., and the HIF system is provided by 2-oxoglutarate (2OG)-dependent N.J.K. performed research; J.S.S. contributed new reagents/analytic tools; J.S.S., I.K.H.L., W.G., and Fe(II)-dependent oxygenases, which catalyze prolyl-4-hydrox- M.A.B., J.P., H.B.K., C.M.T., T.D.W.C., G.M.P., M.A.M., and C.J.S. analyzed data; and J.S.S., S.H., ylation of HIF-α subunits, a posttranslational modification that M.A.M., and C.J.S. wrote the paper. enhances binding of HIF-α to the von Hippel-Lindau protein The authors declare no conflict of interest. (pVHL), so targeting HIF-α for proteasomal degradation. The Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, HIF prolyl-hydroxylases (PHDs) belong to a subfamily of 2OG www.pdb.org (PDB ID codes 4J25, 4J0Q, 4IW3), and sequence data deposited in Uniprot, www.uniprot.org/uniprot (accession nos. Q88CM1 and Q9I6I1) and GenBank, www.ncbi. oxygenases that catalyze prolyl-hydroxylation, which also includes nih.gov/protein (accession nos. AAN70724.1 and AAG03699.1). the collagen prolyl-3-hydroxylases (CP3Hs) and prolyl-4-hydrox- 1I.K.H.L. and W.G. contributed equally to this work. ylases (CP4Hs) (4). Subsequently identified prolyl-hydroxylases 2 Present address: School of Chemical Sciences, University of Auckland, Auckland 1142, include the ribosomal prolyl-hydroxylases (OGFOD1 and Tpa1), New Zealand. which catalyze ribosomal protein 23 prolyl-3-hydroxylation in many 3Present address: Department of Medical Biology, University of Tromsø, N-9019 Tromsø, Norway. eukaryotes, and slime-mold enzymes, which catalyze prolyl-4-hy- 4Present address: Department of Chemistry, University of Tokyo, 113-0033 Tokyo, Japan. – droxylation of Skp1, a ubiquitin ligase subunit (5 9). The HIF- 5Present address: Helmholtz Zentrum Munchen, 85765 Munchen, Germany. PHD-VHL triad is likely present in all animals, but probably 6Present address: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, not in other organisms (3). However, structurally informed Australia. bioinformatic analyses imply the presence of PHD homologs in 7To whom correspondence should be addressed. Email: [email protected]. bacteria (10, 11), including in Pseudomonas spp, suggesting This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. PHDs may have ancient origins. 1073/pnas.1409916111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1409916111 PNAS | September 16, 2014 | vol. 111 | no. 37 | 13331–13336 Downloaded by guest on October 1, 2021 including pyocyanin (13), and causes HIF-α up-regulation via to reflect its role as a hypoxia sensor, these preliminary differ- PHD inhibition, suggesting related roles for PPHD and the animal ences are of interest. In particular, the higher Km for Fe(II) is PHDs (1). notable given the phenotypic results on the PPHD insertional mutant strain, supporting a role for PPHD in iron regulation. Elongation Factor Tu Is a PPHD Substrate. We then investigated The switch I loop of EF-Tu plays a central role in bacterial possible PPHD substrates. Mass spectrometric (MS) studies translation by coupling GTP hydrolysis with conformational identified elongation factor Tu (EF-Tu) as a PPHD binding changes from the GTP-bound active state to the GDP-bound partner and potential substrate in Pseudomonas putida (SI Ap- inactive state (21, 22). NMR was used to directly monitor pendix,TableS3). This assignment is supported by a proteomic kirromycin-induced GTP hydrolysis by EF-Tuputida; we observed MS study on Shewanella oneidensis, reporting that EF-Tu is mod- no difference in the GTP hydrolysis rate for Hyp54 EF-Tu (0.71 ± ified by a +16-Da mass shift (14); bioinformatics reveal Shewanella 0.02 μM/min) and unmodified EF-Tu (0.76 ± 0.08 μM/min; – oneidensis contains a potential hydroxylase closely related to SI Appendix,Fig.S5A). However, addition of PPHDputida to the EF PPHD (38% identity). We tested a set of 14 synthetic peptides Tu GTP hydrolysis reaction decreased the rate of GTP hydrolysis covering the 19 prolines in the EF-Tu sequence as substrates for in a metal ion and 2OG-dependent manner [Zn(II) 0.60 ± 0.05 μ ± μ isolated recombinant PPHDputida (SI Appendix, Table S4). We M/min; Zn(II) and 2OG 0.49 0.16 M/min; Zn(II) was used as identified an EF-Tu peptide as undergoing a +16-Da mass shift a surrogate for Fe(II) and a PPHDputida inhibitor; SI Appendix, – (Fig. 1B and SI Appendix, Table S4); MSMS analyses demon- Fig. S5 B D]. In P. aeruginosa, we observed no difference in the strated hydroxylation at Pro54 (Hyp54; SI Appendix, Fig. S2A). global translation rate between the PPHD mutant strain and the wild-type under standard growth conditions, as measured by Amino acid analyses reveal that PPHDputida catalyzes trans 35 prolyl-4-hydroxylation of EF-Tu, as occurs for PHD-catalyzed [ S]methionine incorporation (SI Appendix,Fig.S5E). The effect of PPHDputida on the GTP hydrolysis rate is less with hydroxy- HIF and CP4H-catalyzed collagen prolyl-hydroxylation (SI Ap- ± μ pendix, Fig.