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Characterization of His-tagged Rat Uroporphyrinogen III Synthase Wild-Type and Variant Enzymes

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Abstract

The structurally related tetrapyrrolic pigments are a group of natural products that participate in many of the fundamental biosynthetic and catabolic processes of living organisms. Urogen III synthase catalyzes a key step in the formation of urogen III, a common intermediate for tetrapyrrolic natural products. In the present study, we cloned, purified, and characterized His-tagged rat urogen III synthase. The mechanism of enzymatic reaction was studied through site-directed mutagenesis of eight highly conserved residues with functional side chains around the active site followed with activity tests. Lys10, Asp17, Glu68, Tyr97, Asn121, Lys147, and His173 have not been studied previously, which were found to be unessential for enzymatic reaction. Tyr168 was identified as an important residue for enzymatic reaction catalyzed by rat urogen III synthase. Molecular modeling suggests the hydroxyl group of Tyr168 side chain is 3.5 Å away from the D ring, and is within hydrogen bond distance (1.9 Å) with acetate side chain of the D ring.

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Abbreviations

CEP:

Congenital erythropoetic porphyria

DTT:

Dithiothreitol

HMB Synthase:

hydroxymethylbilane synthase

IPTG:

Isopropyl-β-D-thiogalactopyranoside

PAGE:

Polyacrylamide gel electrophoresis

PBG:

Porphobilinogen

PCR:

Polymerase chain reaction

SDS:

Sodium dodecylsulfate

urogen III:

Uroporphyrinogen III

urogen III synthase or U3S:

Uroporphyrinogen III synthase

UV/Vis:

Ultraviolet-visible spectroscopy

References

  1. Shoolingin-Jordan PM, Cheung K-M (1999) Compr Nat Prod Chem 4:61–107

    Article  CAS  Google Scholar 

  2. Shoolingin-Jordan PM (1998) Biochem Soc Trans 26:326–336

    CAS  Google Scholar 

  3. Cornah JE, Terry MJ, Smith AG (2003) Trends Plant Sci 8:224–230

    Article  CAS  Google Scholar 

  4. Vavilin DV, Vermaas WFJ (2002) Physiol Plant 115:9–24

    Article  CAS  Google Scholar 

  5. Brusslan JA, Peterson MP (2002) Photosynth Res 71:185–194

    Article  CAS  Google Scholar 

  6. Papenbrock J, Grimm B (2001) Planta 213:667–681

    Article  CAS  Google Scholar 

  7. Moulin M, Smith AG (2005) Biochem Soc Trans 33:737–742

    Article  CAS  Google Scholar 

  8. Grimm B (1998) Curr Opin Plant Biol 1:245–250

    Article  CAS  Google Scholar 

  9. Battersby AR, Leeper FJ (1990) Chem Rev 90:1261–1274

    Article  CAS  Google Scholar 

  10. Tietze LF, Geissler H, Schulz G (1994) Pure Appl Chem 66:2303–2306

    Article  CAS  Google Scholar 

  11. Shady AA, Colby BR, Cunha LF, Astrin KH, Bishop DF, Desnick RJ (2002) Br J Haematol 117:980–987

    Article  CAS  Google Scholar 

  12. Cunha LF, Bishop DF, Desnick RJ (2002) Am J Hum Genet 71:419–419

    Google Scholar 

  13. Bishop DF, Johansson A, Phelps R, Shady AA, Ramirez MCM, Yasuda M, Caro A, Desnick RJ (2006) Am J Hum Genet 78:645–658

    Article  CAS  Google Scholar 

  14. Aizencang GI, Bishop DF, Forrest D, Astrin KH, Desnick RJ (2000) J Biol Chem 275:2295–2304

    Article  CAS  Google Scholar 

  15. Xu WM, Astrin KH, Desnick RJ (1996) Hum Mutat 7:187–192

    Article  CAS  Google Scholar 

  16. Crockett N, Alefounder PR, Battersby AR, Abell C (1991) Tetrahedron 47:6003–6014

    Article  CAS  Google Scholar 

  17. Hanaoka R, Dawid IB, Kawahara A (2007) DNA Seq 18:54–60

    Article  CAS  Google Scholar 

  18. Xu WM, Kozak CA, Desnick RJ (1995) Genomics 26:556–562

    Article  CAS  Google Scholar 

  19. Amillet JM, Labbebois R (1995) Yeast 11:419–424

    Article  CAS  Google Scholar 

  20. Bensidhoum M, Ged CM, Poirier C, Guenet JL, Deverneuil H (1994) Mamm Genome 5:728–730

    Article  CAS  Google Scholar 

  21. Tsai SF, Bishop DF, Desnick RJ (1988) Proc Natl Acad Sci USA 85:7049–7053

    Google Scholar 

  22. Alwan AF, Jordan PM (1988) Biochem Soc Trans 16:965–966

    CAS  Google Scholar 

  23. Stamford NP, Capretta A, Battersby AR (1995) Eur J Biochem 231:236–241

    Article  CAS  Google Scholar 

  24. Smythe E, Williams DC (1988) Biochem J 253:275–279

    CAS  Google Scholar 

  25. Mathews MA, Schubert HL, Whitby FG, Alexander KJ, Schadick K, Bergonia HA, Phillips JD, Hill CP (2001) EMBO J 20:5832–5839

    Article  CAS  Google Scholar 

  26. Schubert HL, Raux E, Matthews MA, Phillips JD, Wilson KS, Hill CP, Warren MJ (2002) Biochem Soc Trans 30:595–600

    Article  CAS  Google Scholar 

  27. Petersen PM, Hawker CJ, Stamford NPJ, Leeper FJ, Battersby AR (1998) J Chem Soc Perkin Trans 1 9:1531–1539

    Article  Google Scholar 

  28. Spivey AC, Capretta A, Frampton CS, Leeper FJ, Battersby AR (1996) J Chem Soc Perkin Trans 1 2091–2102

    Google Scholar 

  29. Cassidy MA, Crockett N, Leeper FJ, Battersby AR (1996) J Chem Soc Perkin Trans 1 17:2079–2090

    Article  Google Scholar 

  30. Spivey AC, Capretta A, Frampton CS, Leeper FJ, Battersby AR (1995) J Chem Soc Chem Commun 1789–1790

  31. Stark WM, Hawker CJ, Hart GJ, Philippides A, Petersen PM, Lewis JD, Leeper FJ, Battersby AR (1993) J Chem Soc Perkin Trans 1 23:2875–2892

    Article  Google Scholar 

  32. Cassidy MA, Crockett N, Leeper FJ, Battersby AR (1991) J Chem Soc Chem Commun 6:384–386

    Google Scholar 

  33. Stark WM, Baker MG, Leeper FJ, Raithby PR, Battersby AR (1988) J Chem Soc Perkin Trans 1 5:1187–1201

    Article  Google Scholar 

  34. Stark WM, Hart GJ, Battersby AR (1986) J Chem Soc Chem Commun 6:465–467

    Google Scholar 

  35. Pichon C, Atshaves BP, Xue TH, Stolowich NJ, Scott AI (1994) Bioorg Med Chem Lett 4:1105–1110

    Article  CAS  Google Scholar 

  36. Roessner CA, Ponnamperuma K, Scott AI (2002) FEBS Lett 525:25–28

    Article  CAS  Google Scholar 

  37. Sodeoka M, Larson CJ, Chen L, LeClair KP, Verdine, Gregory L (1993) Bioorg Med Chem Lett 3:1089–1094

  38. Chu X, Li D (2003) Protein Expr Purif 27:165–170

    Article  CAS  Google Scholar 

  39. Shoolingin-Jordan PM, Leadbeater R (1997) Methods Enzymol 281:327–336

    Article  CAS  Google Scholar 

  40. Totrov M, Abagyan R (1997) Proteins Suppl 1 29:215–220

    Google Scholar 

  41. Levin EY (1971) Biochemistry 10:4669–4675

    Article  CAS  Google Scholar 

  42. Tsai SF, Bishop DF, Desnick RJ (1987) J Biol Chem 262:1268–1273

    CAS  Google Scholar 

  43. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) Nucleic Acids Res 25:4876–4882

    Article  CAS  Google Scholar 

  44. Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003) Nucleic Acids Res 31:3497–3500

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The work described in this paper was financially supported by the City University of Hong Kong.

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Authors and Affiliations

  1. Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, P.R. China

    Nan Li, Xiaojun Liu, Long Wu, Xiusheng Chu & Ding Li

  2. Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, P.R. China

    Dik-Lung Ma & Kwok-yin Wong

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  1. Nan Li
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  2. Dik-Lung Ma
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  3. Xiaojun Liu
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  4. Long Wu
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  7. Ding Li
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Correspondence to Ding Li.

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Li, N., Ma, DL., Liu, X. et al. Characterization of His-tagged Rat Uroporphyrinogen III Synthase Wild-Type and Variant Enzymes. Protein J 26, 569–576 (2007). https://doi.org/10.1007/s10930-007-9099-7

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