Skip to main content
SpringerLink
Log in

Identification of Critical Amino Acid Residues for Human iNOS Functional Activity

  • Published:
The Protein Journal Aims and scope Submit manuscript

Abstract

Nitric oxide (NO) is a short-lived signaling molecule that mediates a variety of biological functions, including vascular homeostasis, neurotransmission, antimicrobial defense and antitumor activities. Three known NOS isoforms (eNOS, nNOS and iNOS) have been cloned and sequenced. Here, we show that upon expression in Escherichia coli using a novel expression vector, an iNOS sequence containing three mutations (A805D, F831S and L832P) within the iNOS reductase domain produced very little functionally active iNOS protein compared to the wild type (wt) iNOS. Each of these point mutations also was individually constructed into the wt iNOS sequence. The activity of the iNOS protein containing the A805D mutation was comparable to wt, while a drastic reduction in iNOS activity was observed for the F831S and L832P mutants. A comparison of the molecular models of the reductase domain of the wt and mutant iNOS revealed a reduced core packing density for the F831S and L832P mutations compared to wt. In addition, the modeling also suggests altered hydrogen bonding, van der Waals and hydrophobic interactions of these mutants.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

NO:

Nitric oxide

NOS:

Nitric oxide synthase

LPS:

Lipopolysaccharide

FAD:

Flavin Adenine Dinucleotide

H4B:

Tetrahydrobiopterin

SMT:

2-Methyl-2-thiopseudourea, sulfate

PBITU:

[S, S′-1,3-Phenylene-bis (1,2-ethanediyl)-bis-isothiourea, 2HBr]

l-NIL:

[l-N6-(1-Iminoethyl)lysine, DiHCl]

1400W:

[N-(3-Aminomethyl)benzylacetamidine, 2HCl]

l-NNA:

N G-Nitro-l-arginine

7-Ni:

7-Nitroindazole

N-PLA:

N G-Propyl-l-arginine

References

  1. Abu-Soud HM, Stuehr DJ (1993) Proc Natl Acad Sci USA 90:10769–10772

    Article  CAS  Google Scholar 

  2. Alderton WK, Cooper CE, Knowles RG (2001) Biochem J 357:593

    Article  CAS  Google Scholar 

  3. Bradford MM (1976) Anal Biochem 72:248

    Article  CAS  Google Scholar 

  4. Brooks BR, Bruccoleri RE, Olafson BD, States DJ, Swaminathan S, Karplus M (1983) J Comp Chem 4:187

    Google Scholar 

  5. Castrillo A, Diaz-Guerra MJ, Hortelano S, Martin-Sanz P, Bosca L (2000) Mol Cell Biol 20:1692

    Article  CAS  Google Scholar 

  6. Charles IG, Palmer RM, Hickery MS, Bayliss MT, Chubb AP, Hall VS, Moss DW, Moncada S (1993) Proc Natl Acad Sci USA 90:11419

    Article  CAS  Google Scholar 

  7. Cho HJ, Xie QW, Calaycay WJ, Mumford RA, Swiderek KM, Lee LD, Nathan C (1992) J Exp Med 176:599

    Article  CAS  Google Scholar 

  8. Strausberg RL, Feingold EA, Grouse LH, Derge DG, Klausner RD et al (2002) Proc Natl Acad Sci USA 99:16899

    Article  Google Scholar 

  9. Fosseta JD, Niu XD, Lunn CA, Zavodny PJ, Narula S (1996) FEBS Lett 379:135

    Article  Google Scholar 

  10. Garber NC, Nishida CR (1997) Arch Biochem Biophys 343:249

    Article  Google Scholar 

  11. Garcin ED, Bruns CM, Lloyd SJ, Hosfield DJ, Tiso M, Gachhui R, Stuehr DJ, Tainer JA, Getzoff ED (2004) J Biol Chem 27:37918

    Article  Google Scholar 

  12. Geller DA, Lowenstein CJ, Shapiro RA, Nussler AK, Di Silvio M, Wang SC, Nakayama DK, Simmons RL, Snyder SH, Billiar TR (1993) Proc Natl Acad Sci USA 90:3491

    Article  CAS  Google Scholar 

  13. Griffith OW, Stuehr DJ (1995) Annu Rev Physiol 57:707

    Article  CAS  Google Scholar 

  14. Jao SC, Huang LF, Hwang SM, Li WS (2006) Biochemistry 45:1547

    Article  CAS  Google Scholar 

  15. Kroncke DJ, Feshel K, Kolb-Bachofen V (1995) Biol Chem Hoppe-Seyler 376:327

    CAS  Google Scholar 

  16. Levy D, Hoke A, Zochodne DW (1999) Neurosci Lett 260:207

    Article  CAS  Google Scholar 

  17. Livingston RJ, Rieder MJ, Shaffer T, Bertucci C, Baier CN et al (2005) GenBank accession AAY43131

  18. Marletta MA (1999) J Biol Chem 268:12231

    Google Scholar 

  19. Nathan CF, Xie QW (1994) J Biol Chem 269:13725

    CAS  Google Scholar 

  20. Palmer RM (1988) Nature 333:664

    Article  CAS  Google Scholar 

  21. Radisky ES, Lu C-JK, Kwan G, Koshland DE Jr (2005) Biochemistry 44:6823

    Article  CAS  Google Scholar 

  22. Sali A, Blundell T (1993) J Mol Biol 234:779

    Article  CAS  Google Scholar 

  23. Sali A, Potterton L, Yuan F, van Vlijmen H, Karplus M (1995) Proteins 23:318

    Article  CAS  Google Scholar 

  24. Sherman PA, Laubach VE, Reep BR, Wood ER (1993) Biochemistry 32:11600

    Article  CAS  Google Scholar 

  25. Sung CS, Wen ZH, Chang WK, Ho ST, Tsai SK, Chang YC, Wong CH (2004) Brain Res 1015:145

    Article  CAS  Google Scholar 

  26. Vallance P, Leiper J (2002) Nat Rev Drug Discov 1:939

    Article  CAS  Google Scholar 

  27. Zimmerman SA, Ferry JG (2006) Biochemistry 45:5149

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to Dr. Brain Bates and Brendan Bingham for critical reading of the manuscript and useful suggestions.

Author information

Author notes

  1. Saule Naureckiene

    Present address: J&J PRD, LLC, 1125 Trenton-Harbourton Road, Titusville, NJ, 08560, USA

  2. Philip G. Jones

    Present address: Sepracor Inc., 84 Waterford Drive, Marlborough, MA, 01752, USA

  3. Kathryn E. Rogers

    Present address: Envivo Pharmaceuticals, 480 Arsenal Street, Watertown, MA, 02472, USA

Authors and Affiliations

  1. Neuroscience Discovery Research, Wyeth Research, CN8000, Princeton, NJ, 08543, USA

    Saule Naureckiene, Edward J. Kaftan, Philip G. Jones, Jeffrey D. Kennedy, Kathryn E. Rogers & Pranab K. Chanda

  2. Biological Technologies, Wyeth Research, CN8000, Princeton, NJ, 08543, USA

    Sreekumar R. Kodangattil

Authors
  1. Saule Naureckiene
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sreekumar R. Kodangattil
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Edward J. Kaftan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Philip G. Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jeffrey D. Kennedy
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kathryn E. Rogers
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Pranab K. Chanda
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Pranab K. Chanda.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Naureckiene, S., Kodangattil, S.R., Kaftan, E.J. et al. Identification of Critical Amino Acid Residues for Human iNOS Functional Activity. Protein J 27, 309–318 (2008). https://doi.org/10.1007/s10930-008-9139-y

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10930-008-9139-y

Keywords

Search

Navigation