Journal of Molecular Evolution

, Volume 86, Issue 7, pp 457–469 | Cite as

Sequential Molecular Events of Functional Trade-Offs in 5-Hydroxyisourate Hydrolase Before and After Gene Duplication Led to the Evolution of Transthyretin During Chordate Diversification

  • Kiyoshi Yamauchi
  • Kentaro Kasai
Original Article


Transthyretin (TTR), a plasma thyroid hormone distributor protein (THDP), emerged from 5-hydroxyisourate hydrolase (HIUHase), an enzyme involved in urate metabolism, by gene duplication at a stage of chordate evolution. Comparison of amino acid sequences revealed the presence of two His-rich segments in the primitive TTRs. Using several HIUHase and TTR mutants, we investigated 5-hydroxyisourate (HIU) hydrolysis activity and thyroid hormone (TH) binding activity to elucidate how a novel function as a THDP arose. Lancelet HIUHase was found to have higher enzyme activity than trout HIUHase. Two amino acid substitutions, R54E/Y119T, at the active sites of HIUHase, exerted weak [125I]-3,3′,5-triiodo-l-thyronine ([125I]T3) binding activity with a concomitant loss of HIU hydrolysis activity. Addition of 3×His (3×H) to the N-terminal end weakened HIU hydrolysis activity of both lancelet and trout HIUHases, whereas it enhanced T3-binding activity of HIUHase R54E/Y119T. Trout HIUHase 3×H R54E/Y119T had higher [125I]T3-binding activity than that of lancelet HIUHase 3×H R54E/Y119T, with a Kd of 143 nM, and displayed metal dependency and no TH binding specificity. Deletion of the N-terminal His-rich segment from lamprey TTR decreased T3-binding activity, while addition of 3×H to trout TTR increased T3-binding activity, while maintaining TH binding specificity. Our results suggest that functional trade-offs of HIU hydrolysis activity with TH binding activity might have sequentially occurred before and after gene duplication, and that TH binding specificity and high-affinity sites may have been acquired later in the course of TTR evolution.


Transthyretin Thyroid hormone Hydroxyisourate hydrolases Functional trade-off Evolution 



Bovine serum albumin


Coomassie brilliant blue


Ethylenediaminetetraacetic acid


Hydrophobic interaction column chromatography




5-Hydroxyisourate hydrolase


High-performance liquid chromatography


Dissociation constant


Maximum binding capacity


Polyacrylamide-gel electrophoresis


Polymerase chain reaction


Rapid amplification of cDNA end


Reverse T3 or 3,3′,5′-triiodo-l-thyronine


Reverse transcription


Sodium dodecyl sulfate








Tris-buffered saline


Thyroid hormone


TH binding protein


3,3′,5-Triiodothyroacetic acid


3,3′,5,5′-Tetraiodothyroacetic acid





We are grateful to Akihiro Ito, Shunsuke Kawamoto, Taishi Aoki, Shunsuke Suzuki, Syuko Sakai, and Yuki Ota for their technical assistance in recombinant protein preparation and hormone binding assays. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Cendron L, Ramazzina I, Percudani R, Rasore C, Zanotti G, Berni R (2011) Probing the evolution of hydroxyisourate hydrolase into transthyretin through active-site redesign. J Mol Biol 409:504–512. CrossRefPubMedGoogle Scholar
  2. Conant GC, Wolfe KH (2008) Turning a hobby into a job: how duplicated genes find new functions. Nat Rev Genet 9:938–950. CrossRefPubMedGoogle Scholar
  3. Deng C, Cheng CH, Ye H, He X, Chen L (2010) Evolution of an antifreeze protein by neofunctionalization under escape from adaptive conflict. Proc Natl Acad Sci USA 107:21593–21598. CrossRefPubMedGoogle Scholar
  4. Des Marais DL, Rausher MD (2008) Escape from adaptive conflict after duplication in an anthocyanin pathway gene. Nature 454:762–765. CrossRefPubMedGoogle Scholar
  5. Eales JG (1997) Iodine metabolism and thyroid-related functions in organisms lacking thyroid follicles: are thyroid hormones also vitamins? Proc Soc Exp Biol Med 214:302–317CrossRefPubMedGoogle Scholar
  6. Eneqvist T, Lundberg E, Nilsson L, Abagyan R, Sauer-Eriksson AE (2003) The transthyretin-related protein family. Eur J Biochem 270:518–532CrossRefPubMedGoogle Scholar
  7. Eneqvist T, Lundberg E, Karlsson A, Huang S, Santos CR, Power DM, Sauer-Eriksson AE (2004) High resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxine. J Biol Chem 279:26411–26416. CrossRefPubMedGoogle Scholar
  8. Folli C, Pasquato N, Ramazzina I, Battistutta R, Zanotti G, Berni R (2003) Distinctive binding and structural properties of piscine transthyretin. FEBS Lett 555:279–284CrossRefPubMedGoogle Scholar
  9. Hennebry SC, Law RH, Richardson SJ, Buckle AM, Whisstock JC (2006a) The crystal structure of the transthyretin-like protein from Salmonella dublin, a prokaryote 5-hydroxyisourate hydrolase. J Mol Biol 359:1389–1399. CrossRefPubMedGoogle Scholar
  10. Hennebry SC, Wright HM, Likic VA, Richardson SJ (2006b) Structural and functional evolution of transthyretin and transthyretin-like proteins. Proteins 64:1024–1045. CrossRefPubMedGoogle Scholar
  11. Heyland A, Hodin J, Reitzel AM (2005) Hormone signaling in evolution and development: a non-model system approach. Bioessays 27:64–75. CrossRefPubMedGoogle Scholar
  12. Jain S, Sharma S, Gupta MN (2002) A microassay for protein determination using microwaves. Anal Biochem 311:84–86CrossRefPubMedGoogle Scholar
  13. Jung DK, Lee Y, Park SG, Park BC, Kim GH, Rhee S (2006) Structural and functional analysis of PucM, a hydrolase in the ureide pathway and a member of the transthyretin-related protein family. Proc Natl Acad Sci USA 103:9790–9795. CrossRefPubMedGoogle Scholar
  14. Kasai K, Nishiyama N, Yamauchi K (2013) Characterization of Oncorhynchus mykiss 5-hydroxyisourate hydrolase/transthyretin superfamily: evolutionary and functional analyses. Gene 531:326–336. CrossRefPubMedGoogle Scholar
  15. Kasai K, Nishiyama N, Yamauchi K (2018) Molecular and thyroid hormone binding properties of lamprey transthyretins: the role of an N-terminal histidine-rich segment in hormone binding with high affinity. Mol Cell Endocrinol. PubMedCrossRefGoogle Scholar
  16. Kurochkin IV, Mizuno Y, Konagaya A, Sakaki Y, Schönbach C, Okazaki Y (2007) Novel peroxisomal protease Tysnd1 processes PTS1- and PTS2-containing enzymes involved in beta-oxidation of fatty acids. EMBO J 26:835–845. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Lee Y, Lee DH, Kho CW, Lee AY, Jang M, Cho S, Lee CH, Lee JS, Myung PK, Park BC, Park SG (2005) Transthyretin-related proteins function to facilitate the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction. FEBS Lett 579:4769–4774. CrossRefPubMedGoogle Scholar
  18. Li Z, Yao F, Li M, Zhang S (2013) Identification and bioactivity analysis of transthyretin-like protein in amphioxus: a case demonstrating divergent evolution from an enzyme to a hormone distributor. Comp Biochem Physiol B 164:143–150. CrossRefPubMedGoogle Scholar
  19. Liz MA, Leite SC, Juliano L, Saraiva MJ, Damas AM, Bur D, Sousa MM (2012) Transthyretin is a metallopeptidase with an inducible active site. Biochem J 443:769–778. CrossRefPubMedGoogle Scholar
  20. Lundberg E, Bäckström S, Sauer UH, Sauer-Eriksson AE (2006) The transthyretin-related protein: structural investigation of a novel protein family. J Struct Biol 155:445–457. CrossRefPubMedGoogle Scholar
  21. Palmieri LC, Lima LM, Freire JB, Bleicher L, Polikarpov I, Almeida FC, Foguel D (2010) Novel Zn2+-binding sites in human transthyretin: implications for amyloidogenesis and retinol-binding protein recognition. J Biol Chem 285:31731–31741. CrossRefGoogle Scholar
  22. Paris M, Brunet F, Markov GV, Schubert M, Laudet V (2008) The amphioxus genome enlightens the evolution of the thyroid hormone signaling pathway. Dev Genes Evol 218:667–680. CrossRefPubMedGoogle Scholar
  23. Ramazzina I, Folli C, Secchi A, Berni R, Percudani R (2006) Completing the uric acid degradation pathway through phylogenetic comparison of whole genomes. Nat Chem Biol 2:144–148. CrossRefPubMedGoogle Scholar
  24. Scatchard G (1949) The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51:660–672. CrossRefGoogle Scholar
  25. Schreiber G, Richardson SJ (1997) The evolution of gene expression, structure and function of transthyretin. Comp Biochem Physiol B 116:137–160CrossRefPubMedGoogle Scholar
  26. Sherwood NM, Adams BA, Tello JA (2005) Endocrinology of protochordates. Can J Zool 83:225–255. CrossRefGoogle Scholar
  27. Suzuki S, Kasai K, Yamauchi K (2015) Characterization of little skate (Leucoraja erinacea) recombinant transthyretin: zinc-dependent 3,3′,5-triiodo-l-thyronine binding. Gen Comp Endocrinol 217–218:43–53. CrossRefPubMedGoogle Scholar
  28. Suzuki S, Kasai K, Nishiyama N, Ishihara A, Yamauchi K (2017) Characteristics of the brown hagfish Paramyxine atami transthyretin: metal ion-dependent thyroid hormone binding. Gen Comp Endocrinol 249:1–14. CrossRefPubMedGoogle Scholar
  29. Vandepoele K, De Vos W, Taylor JS, Meyer A, Van de Peer Y (2004) Major events in the genome evolution of vertebrates: paranome age and size differ considerably between ray-finned fishes and land vertebrates. Proc Natl Acad Sci USA 101:1638–1643. CrossRefPubMedGoogle Scholar
  30. Vos MJ, Kampinga HH (2008) A PCR amplification strategy for unrestricted generation of chimeric genes. Anal Biochem 380:338–340. CrossRefPubMedGoogle Scholar
  31. Yamauchi K, Kasahara T, Hayashi H, Horiuchi R (1993) Purification and characterization of a 3,5,3′-l-triiodothyronine-specific binding protein from bullfrog tadpole plasma: a homolog of mammalian transthyretin. Endocrinology 132:2254–2261. CrossRefPubMedGoogle Scholar
  32. Zanotti G, Cendron L, Ramazzina I, Folli C, Percudani R, Berni R (2006) Structure of zebra fish HIUase: insights into evolution of an enzyme to a hormone transporter. J Mol Biol 363:1–9. CrossRefPubMedGoogle Scholar
  33. Zanotti G, Ramazzina I, Cendron L, Folli C, Percudani R, Berni R (2009) Vertebrate 5-hydroxyisourate hydrolase identification, function, structure, and evolutionary relationship with transthyretin. In: Richardson SJ, Cody V (eds) Recent advances in transthyretin evolution, structure and biological functions. Springer, Berlin, pp 95–108. CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biological Science, Graduate School of ScienceShizuoka UniversityShizuokaJapan

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