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Analysis of raphidophyte assimilatory nitrate reductase reveals unique domain architecture incorporating a 2/2 hemoglobin

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Abstract

Eukaryotic assimilatory nitrate reductase (NR) is a multi-domain protein that catalyzes the rate-limiting step in nitrate assimilation. This protein is highly conserved and has been extensively characterized in plants and algae. Here, we report hybrid NRs (NR2-2/2HbN) identified in two microalgal species, Heterosigma akashiwo and Chattonella subsalsa, with a 2/2 hemoglobin (2/2Hb) inserted into the hinge 2 region of a prototypical NR. 2/2Hbs are a class of single-domain heme proteins found in bacteria, ciliates, algae and plants. Sequence analysis indicates that the C-terminal FAD/NADH reductase domain of NR2-2/2HbN retains identity with eukaryotic NR, suggesting that the 2/2Hb domain was inserted interior to the existing NR domain architecture. Phylogenetic analysis supports the placement of the 2/2Hb domain of NR2-2/2HbN within group I (N-type) 2/2Hbs with high similarity to mycobacterial 2/2HbNs, known to convert nitric oxide to nitrate. Experimental data confirms that H. akashiwo is capable of metabolizing nitric oxide and shows that HaNR2-2/2HbN expression increases in response to nitric oxide addition. Here, we propose a mechanism for the dual function of NR2-2/2HbN in which nitrate reduction and nitric oxide dioxygenase reactions are cooperative, such that conversion of nitric oxide to nitrate is followed by reduction of nitrate for assimilation as cellular nitrogen.

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References

  • Allen AE, Ward BB, Song B (2005) Characterization of diatom (Bacillariophyceae) nitrate reductase genes and their detection in marine phytoplankton communities. J Phycol 41:95–104

    Article  CAS  Google Scholar 

  • Altschul SF et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl Acids Res 25:3389–3402

    Article  PubMed  CAS  Google Scholar 

  • Armbrust EV et al (2004) The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306:79–86

    Article  PubMed  CAS  Google Scholar 

  • Baldauf SL (2003) The deep roots of eukaryotes. Science 300:1703–1706

    Article  PubMed  CAS  Google Scholar 

  • Baldauf SL (2008) An overview of the phylogeny and diversity of eukaryotes. J Syst Evol 46:263–273

    Google Scholar 

  • Berges J (1997) Miniview: algal nitrate reductases. Eur J Phycol 32:3–8

    Article  Google Scholar 

  • Bjorklund AK, Ekman D, Light S, Frey-Skott J, Elofsson A (2005) Domain rearrangements in protein evolution. J Mol Biol 353:911–923

    Article  PubMed  Google Scholar 

  • Bonamore A et al (2007) A novel chimera: the “truncated hemoglobin-antibiotic monooxygenase” from Streptomyces avermitilis. Gene 398:52–61

    Article  PubMed  CAS  Google Scholar 

  • Bowler C et al (2008) The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456:239–244

    Article  PubMed  CAS  Google Scholar 

  • Campbell WH (1999) Nitrate reductase structure, function and regulation: bridging the gap between biochemistry and physiology. Annu Rev Plant Physiol Plant Mol Biol 50:277–303

    Article  PubMed  CAS  Google Scholar 

  • Campbell WH (2001) Structure and function of eukaryotic NAD(P)H:nitrate reductase. Cell Mol Life Sci 58:194–204

    Article  PubMed  CAS  Google Scholar 

  • Chenna R (2003) Multiple sequence alignment with the clustal series of programs. Nucl Acids Res 31:3497–3500

    Article  PubMed  CAS  Google Scholar 

  • Coyne KJ (2010) Nitrate reductase (NR1) sequence and expression in the harmful alga Heterosigma akashiwo (Raphidophyceae). J Phycol 46:135–142

    Article  CAS  Google Scholar 

  • Coyne KJ, Cary SC (2005) Molecular approaches to the investigation of viable dinoflagellate cysts in natural sediments from estuarine environments. J Eukaryot Microbiol 52:90–94

    Article  PubMed  CAS  Google Scholar 

  • Coyne K, Hutchins D, Hare C, Cary S (2001) Assessing temporal and spatial variability in Pfiesteria piscicida distributions using molecular probing techniques. Aquat Microb Ecol 24:275–285

    Article  Google Scholar 

  • Coyne KJ, Burkholder JM, Feldman RA, Hutchins DA, Cary SC (2004) Modified serial analysis of gene expression method for construction of gene expression profiles of microbial eukaryotic species. Appl Environ Microbiol 70:5298–5304

    Article  PubMed  CAS  Google Scholar 

  • Crawford NM (2006) Mechanisms for nitric oxide synthesis in plants. J Exp Bot 57(3):471–478

    Article  PubMed  CAS  Google Scholar 

  • Desikan R, Griffiths R, Hancock J, Neill S (2002) A new role for an old enzyme: nitrate reductase mediated nitric oxide generation is required for abscisic acid-induced stomatal closure in Arabidopsis thaliana. PNAS 99(25)16314–16318

    Article  PubMed  CAS  Google Scholar 

  • Doerks T, Copley RR, Shultz J, Ponting CP, Bork P (2002) Systematic identification of novel protein domain families associated with nuclear functions. Genome Res 12:47–56

    Article  PubMed  CAS  Google Scholar 

  • Du S, Zhang Y, Lin X, Wang Y, Tang C (2008) Regulation of nitrate reductase by nitric oxide in Chinese cabbage pakchoi (Brassica chinensis L.) Plant Cell Environ 31:195–204

    Google Scholar 

  • Eckardt NA (2005) Moco Mojo: crystal structure reveals essential features of eukaryotic assimilatory nitrate reduction. Plant Cell 17:1029–1031

    Article  CAS  Google Scholar 

  • Farlow A, Meduri E, Schlötterer C (2010) DNA double-strand break repair and the evolution of intron density. Trends Genet 27:1–6

    Article  PubMed  Google Scholar 

  • Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791

    Article  Google Scholar 

  • Gardner PR (2005) Nitric oxide dioxygenase function and mechanism of flavohemoglobin, hemoglobin, myoglobin and their associated reductases. J Inorg Biochem 99:247–266

    Article  PubMed  CAS  Google Scholar 

  • Gardner PR et al (2000) Nitric-oxide dioxygenase activity and function of flavohemoglobins. Sensitivity to nitric oxide and carbon monoxide inhibition. J Biol Chem 275:31581–31587

    Article  PubMed  CAS  Google Scholar 

  • Guillard RRL (1975) Culture of phytoplankton for feeding marine invertebrates. In: Smith WL, Chanley MH (eds) Culture of marine invertebrate animals. Plenum Press, New York, pp 26–60

    Google Scholar 

  • Hacker J, Kaper JB (2000) Pathogenicity islands and the evolution of microbes. Annu Rev Microbiol 54:641–679

    Article  PubMed  CAS  Google Scholar 

  • Hammer Ø, Harper DAT, Ryan PD (2001) PAST: paleontological statistics software package for education and data analysis. Palaeontologia Electronica 4:1–9

    Google Scholar 

  • Handy SM et al (2005) Evaluating vertical migration behavior of harmful raphidophytes in the Delaware Inland Bays utilizing quantitative real-time PCR. Aquat Microb Ecol 40:121–132

    Article  Google Scholar 

  • Handy SM et al (2008) Using quantitative real-time PCR to study competition and community dynamis among Delaware Inland Bays harmful algae in field and laboratory studies. Harmful Algae 7:599–613

    Article  CAS  Google Scholar 

  • Karplus PA, Bruns CM (1994) Structure-function relations for ferredoxin reductase. J Bioenerg Biomembr 26:89–99

    Article  PubMed  CAS  Google Scholar 

  • Kim D (2006) Nitric oxide synthase-like enzyme mediated nitric oxide generation by harmful red tide phytoplankton, Chattonella marina. J Plankton Res 28:613–620

    Article  CAS  Google Scholar 

  • Kim D et al (2008) Detection of nitric oxide (NO) in marine phytoplankters. J Biosci Bioeng 105:414–417

    Article  PubMed  CAS  Google Scholar 

  • Lama A, Pawaria S, Dikshit KL (2006) Oxygen binding and NO scavenging properties of truncated hemoglobin, HbN, of Mycobacterium smegmatis. FEBS Lett 580:4031–4041

    Article  PubMed  CAS  Google Scholar 

  • Lama A et al (2009) Role of pre-A motif in nitric oxide scavenging by truncated hemoglobin, HbN, of Mycobacterium tuberculosis. J Biol Chem 284:14457–14468

    Article  PubMed  CAS  Google Scholar 

  • Landsberg JH (2002) The effects of harmful algal blooms on aquatic organisms. Rev Fish Sci 10:113–390

    Article  Google Scholar 

  • Larkin MA et al (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948

    Article  PubMed  CAS  Google Scholar 

  • Lecomte JTJ, Vuletich DA, Lesk AM (2005) Structural divergence and distant relationships in proteins: evolution of the globins. Curr Opin Struct Biol 15:290–301

    Article  PubMed  CAS  Google Scholar 

  • Milani M, Pesce A, Ouellet H, Guertin M, Bolognesi M (2003) Truncated hemoglobins and nitric oxide action. IUBMB Life 55:623–627

    Article  PubMed  CAS  Google Scholar 

  • Millett F, Durham B (2004) Kinetics of electron transfer within cytochrome bc1 and between cytochrome bc1 and cytochrome c. Photosynth Res 82:1–16

    Article  PubMed  CAS  Google Scholar 

  • Moore AD, Björklund AK, Ekman D, Bornberg-Bauer E, Elofsson A (2008) Arrangements in the modular evolution of proteins. Trends Biochem Sci 33:444–451

    Article  PubMed  CAS  Google Scholar 

  • Mukai M, Mills CE, Poole RK, Yeh SR (2001) Flavohemoglobin, a globin with a peroxidase-like catalytic site. J Biol Chem 276:7272–7277

    Article  PubMed  CAS  Google Scholar 

  • Nardini M, Pesce A, Milani M, Bolognesi M (2007) Protein fold and structure in the truncated (2/2) globin family. Gene 398:2–11

    Article  PubMed  CAS  Google Scholar 

  • Oda T, Nakamura A, Okamoto T, Ishimatsu A, Muramatsu T (1998) Lectin-induced enhancement of superoxide anion production by red tide phytoplankton. Mar Biol 131:383–390

    Article  CAS  Google Scholar 

  • Ouellet H et al (2002) Truncated hemoglobin HbN protects Mycobacterium bovis from nitric oxide. Proc Natl Acad Sci USA 99:5902–5907

    Article  PubMed  CAS  Google Scholar 

  • Padmakumar KB, Thomas LC, Salini TC, John E, Menon NR, Sanjeevan VN (2011) Monospecific bloom of noxious raphidophyte Chattonella marina in the coastal water of South West coast of India. Int J Biosci 1:57–59

    Google Scholar 

  • Poole RK, Hughes MN (2000) New functions for the ancient globin family: bacterial responses to nitric oxide and nitrosative stress. Mol Microbiol 36:775–783

    Article  PubMed  CAS  Google Scholar 

  • Rzhetsky A, Nei M (1992) Statistical properties of the ordinary least-squares, generalized least-squares, and minimum-evolution methods of phylogenetic inference. J Mol Evol 35:367–375

    Article  PubMed  CAS  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    PubMed  CAS  Google Scholar 

  • Sakihama Y, Nakamura S, Yamasaki H (2002) Nitric oxide production mediated by nitrate reductase in the green alga Chlamydomonas reinhardtii: an alternative NO production pathway in photosynthetic organisms. Plant Cell Physiol 43:290–297

    Article  PubMed  CAS  Google Scholar 

  • Selvam RA, Sasidharan R (2004) DomIns: a web resource for domain insertions in known protein structures. Nucl Acids Res 32:D193–D195

    Article  PubMed  CAS  Google Scholar 

  • Smagghe BJ, Trent JT, Hargrove MS (2008) NO dioxygenase activity in hemoglobins is ubiquitous in vitro, but limited by reduction in vivo. PloS One 3:e2039

    Article  PubMed  Google Scholar 

  • Stolz JF, Basu P (2002) Evolution of nitrate reductase: molecular and structural variations on a common function. Chembiochem 3:198–206

    Article  PubMed  CAS  Google Scholar 

  • Szabo C, Ischiropoulos H, Radi R (2007) Peroynitrite: biochemistry, pathophysiology and development of therapeutics. Nat Rev Drug Discov 6:662–680

    Article  PubMed  CAS  Google Scholar 

  • Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    Article  PubMed  CAS  Google Scholar 

  • Taylor FJR, Haigh R (1993) The ecology of fish-killing blooms of the chloromonad flagellate Heterosigma in the Strait of Georgia and adjacent waters. In: Smayda TJ, Shimizu Y (eds) Toxic phytoplankton blooms in the sea. Elsevier Science Publishers, pp 705–710

  • Vardi A et al (2006) A stress surveillance system based on calcium and nitric oxide in marine diatoms. PLoS Biol 4(3):e60

    Article  PubMed  Google Scholar 

  • Vardi A, Thamatrakoln K, Bidle KD, Falkowski PG (2008) Diatom genomes come of age. Genome Biol 9:245

    Article  PubMed  Google Scholar 

  • Vinogradov SN, Moens L (2008) Diversity of globin function: enzymatic, transport, storage, and sensing. J Biol Chem 283:8773–8777

    Article  PubMed  CAS  Google Scholar 

  • Vinogradov SN et al (2005) Three globin lineages belonging to two structural classes in genomes from the three kingdoms of life. Proc Natl Acad Sci USA 102:11385–11389

    Article  PubMed  CAS  Google Scholar 

  • Vinogradov SN et al (2007) A model of globin evolution. Gene 398:132–142

    Article  PubMed  CAS  Google Scholar 

  • Vuletich DA, Lecomte JTJ (2006) A phylogenetic and structural analysis of truncated hemoglobins. J Mol Evol 62:196–210

    Article  PubMed  CAS  Google Scholar 

  • Weiner J 3rd, Beaussart F, Bornberg-Bauer E (2006) Domain deletions and substitutions in the modular protein evolution. FEBS J 273:2037–2047

    Article  PubMed  CAS  Google Scholar 

  • Wittenberg J, Bolognesi M, Wittenberg B, Guertin M (2002) Truncated hemoglobins: a new family of hemoglobins widely distributed in bacteria, unicellular eukaryotes, and plants. J Biol Chem 277:871–874

    Article  PubMed  CAS  Google Scholar 

  • Workun GJ, Moquin K, Rothery RA, Weiner JH (2008) Evolutionary persistence of the molybdopyranopterin-containing sulfite oxidase protein fold. Microbiol Mol Biol Rev 72:228–248

    Article  PubMed  CAS  Google Scholar 

  • Xing L, Zhang Z-B, Liu C-Y, Wu Z–Z, Lin C (2005) Amperometric detection of nitric oxide with microsensor in the medium of seawater and its applications. Sensors 5:537–545

    Article  CAS  Google Scholar 

  • Yamasaki H, Sakihama Y (2000) Simultaneous production of nitric oxide and peroxynitrite by plant nitrate reductase: in vitro evidence for the NR-dependent formation of active nitrogen species. FEBS Lett 468:89–92

    Article  PubMed  CAS  Google Scholar 

  • Yeh SR, Couture M, Ouellet Y, Guertin M, Rousseau DL (2000) A cooperative oxygen binding hemoglobin from Mycobacterium tuberculosis. Stabilization of heme ligands by a distal tyrosine residue. J Biol Chem 275:1679–1684

    Article  PubMed  CAS  Google Scholar 

  • Zhang Z, Liu C, Wu Z, Xing L, Li P (2006) Detection of nitric oxide in culture media and studies of nitric oxide formation by marine microalgae. Med Sci Monit 12:75–86

    Google Scholar 

  • Zhou J, Kleinhofs A (1996) Molecular evolution of nitrate reductase genes. J Mol Evol 42:432–442

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Science Foundation (IOS-0745102) and the Environmental Protection Agency (STAR-ECOHAB-R83-3221). We would like to thank Lauren Salvitti, Mark Warner, and Tom Hanson (University of Delaware), and Don Stewart for technical and editorial support.

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  1. University of Delaware College of Earth, Ocean, and Environment, 700 Pilottown Road, Lewes, DE, 19958, USA

    Jennifer J. Stewart & Kathryn J. Coyne

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Correspondence to Kathryn J. Coyne.

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Stewart, J.J., Coyne, K.J. Analysis of raphidophyte assimilatory nitrate reductase reveals unique domain architecture incorporating a 2/2 hemoglobin. Plant Mol Biol 77, 565–575 (2011). https://doi.org/10.1007/s11103-011-9831-8

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