JBIC Journal of Biological Inorganic Chemistry

, Volume 21, Issue 7, pp 865–873 | Cite as

Biosynthesis of Salmonella enterica [NiFe]-hydrogenase-5: probing the roles of system-specific accessory proteins

  • Lisa Bowman
  • Jonathan Balbach
  • Julia Walton
  • Frank Sargent
  • Alison Parkin
Original Paper


A subset of bacterial [NiFe]-hydrogenases have been shown to be capable of activating dihydrogen-catalysis under aerobic conditions; however, it remains relatively unclear how the assembly and activation of these enzymes is carried out in the presence of air. Acquiring this knowledge is important if a generic method for achieving production of O2-resistant [NiFe]-hydrogenases within heterologous hosts is to be developed. Salmonella enterica serovar Typhimurium synthesizes the [NiFe]-hydrogenase-5 (Hyd-5) enzyme under aerobic conditions. As well as structural genes, the Hyd-5 operon also contains several accessory genes that are predicted to be involved in different stages of biosynthesis of the enzyme. In this work, deletions in the hydF, hydG, and hydH genes have been constructed. The hydF gene encodes a protein related to Ralstonia eutropha HoxO, which is known to interact with the small subunit of a [NiFe]-hydrogenase. HydG is predicted to be a fusion of the R. eutropha HoxQ and HoxR proteins, both of which have been implicated in the biosynthesis of an O2-tolerant hydrogenase, and HydH is a homologue of R. eutropha HoxV, which is a scaffold for [NiFe] cofactor assembly. It is shown here that HydG and HydH play essential roles in Hyd-5 biosynthesis. Hyd-5 can be isolated and characterized from a ΔhydF strain, indicating that HydF may not play the same vital role as the orthologous HoxO. This study, therefore, emphasises differences that can be observed when comparing the function of hydrogenase maturases in different biological systems.


Biosynthesis Electrochemistry Hydrogenase Iron–sulfur cluster Metallocenter assembly 









Methylene blue


Membrane bound hydrogenase(s)

Rh. leguminosarum

Rhizobium leguminosarum

R. eutropha

Ralstonia eutropha



This work was supported in part by EPSRC Grant EP/K031589/1 (AP) and by BBSRC via a Targeted Priority Studentship [Grant Number BB/G016690/1] (FS). We thank David Lloyd for assistance in developing the glove-box methylene blue assay method and Aaron Barnes for conducting preliminary electrochemistry experiments.

Supplementary material

775_2016_1385_MOESM1_ESM.pdf (635 kb)
Supplementary material 1 (PDF 635 kb)


  1. 1.
    Stiebritz MT, Reiher M (2012) Chem Sci 3:1739–1751CrossRefGoogle Scholar
  2. 2.
    Parkin A, Bowman L, Roessler MM, Davies RA, Palmer T, Armstrong FA, Sargent F (2012) FEBS Lett 586:536–544CrossRefPubMedGoogle Scholar
  3. 3.
    Zbell AL, Benoit SL, Maier RJ (2007) Microbiology 153:3508–3516CrossRefPubMedGoogle Scholar
  4. 4.
    Goris T, Wait AF, Saggu M, Fritsch J, Heidary N, Stein M, Zebger I, Lendzian F, Armstrong FA, Friedrich B, Lenz O (2011) Nat Chem Biol 7:310–318CrossRefPubMedGoogle Scholar
  5. 5.
    Lukey MJ, Roessler MM, Parkin A, Evans RM, Davies RA, Lenz O, Friedrich B, Sargent F, Armstrong FA (2011) J Am Chem Soc 133:16881–16892CrossRefPubMedGoogle Scholar
  6. 6.
    Pandelia M-E, Fourmond V, Tron-Infossi P, Lojou E, Bertrand P, Léger C, Giudici-Orticoni M-T, Lubitz W (2010) J Am Chem Soc 132:6991–7004CrossRefPubMedGoogle Scholar
  7. 7.
    Shomura Y, Yoon K-S, Nishihara H, Higuchi Y (2011) Nature 479:253–256CrossRefPubMedGoogle Scholar
  8. 8.
    Parkin A, Sargent F (2012) Curr Opin Chem Biol 16:26–34CrossRefPubMedGoogle Scholar
  9. 9.
    Fritsch J, Lenz O, Friedrich B (2013) Nat Rev Microbiol 11:106–114CrossRefPubMedGoogle Scholar
  10. 10.
    Stripp ST, Soboh B, Lindenstrauss U, Braussemann M, Herzberg M, Nies DH, Sawers RG, Heberle J (2013) Biochemistry 52:3289–3296CrossRefPubMedGoogle Scholar
  11. 11.
    Stripp ST, Lindenstrauss U, Sawers RG, Soboh B (2015) PLoS One 10:e0133118CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Stripp ST, Lindenstrauss U, Granich C, Sawers RG, Soboh B (2014) PLoS One 9:e107488CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Dubini A, Sargent F (2003) FEBS Lett 549:141–146CrossRefPubMedGoogle Scholar
  14. 14.
    Fritsch J, Lenz O, Friedrich B (2011) J Bacteriol 193:2487–2497CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Schubert T, Lenz O, Krause E, Volkmer R, Friedrich B (2007) Mol Microbiol 66:453–467CrossRefPubMedGoogle Scholar
  16. 16.
    Fritsch J, Siebert E, Priebe J, Zebger I, Lendzian F, Teutloff C, Friedrich B, Lenz O (2014) J Biol Chem 289:7982–7993CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Forzi L, Sawers RG (2007) Biometals 20:565–578CrossRefPubMedGoogle Scholar
  18. 18.
    Pinske C, Sawers RG (2012) PLoS One 7:e31755CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Albareda M, Pacios LF, Manyani H, Rey L, Brito B, Imperial J, Ruiz-Argüeso T, Palacios JM (2014) J Biol Chem 289:21217–21229CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Manyani H, Rey L, Palacios JM, Imperial J, Ruiz-Argüeso T (2005) J Bacteriol 187:7018–7026CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Rey L, Hidalgo E, Palacios J, Ruiz-Argüeso T (1992) J Mol Biol 228:998–1002CrossRefPubMedGoogle Scholar
  22. 22.
    Lamichhane-Khadka R, Benoit SL, Miller-Parks EF, Maier RJ (2015) Infect Immun 83:311–316CrossRefPubMedGoogle Scholar
  23. 23.
    Bowman L, Flanagan L, Fyfe Paul K, Parkin A, Hunter William N, Sargent F (2014) Biochem J 458:449–458CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Lam LH, Monack DM (2014) PLoS Pathog 10:e1004527CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Maier L, Vyas R, Cordova Carmen D, Lindsay H, Schmidt Thomas Sebastian B, Brugiroux S, Periaswamy B, Bauer R, Sturm A, Schreiber F, von Mering C, Robinson Mark D, Stecher B, Hardt W-D (2013) Cell Host Microbe 14:641–651CrossRefPubMedGoogle Scholar
  26. 26.
    Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, Heger A, Hetherington K, Holm L, Mistry J, Sonnhammer ELL, Tate J, Punta M (2014) Nucleic Acids Res 42:D222–D230CrossRefPubMedGoogle Scholar
  27. 27.
    Bernhard M, Schwartz E, Rietdorf J, Friedrich B (1996) J Bacteriol 178:4522–4529PubMedPubMedCentralGoogle Scholar
  28. 28.
    Albareda M, Manyani H, Imperial J, Brito B, Ruiz-Argüeso T, Böck A, Palacios J-M (2012) BMC Microbiol 12:1–13CrossRefGoogle Scholar
  29. 29.
    Fritsche E, Paschos A, Beisel H-G, Böck A, Huber R (1999) J Mol Biol 288:989–998CrossRefPubMedGoogle Scholar
  30. 30.
    Thomas C, Muhr E, Sawers RG (2015) J Bacteriol 197:2989–2998CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Ludwig M, Schubert T, Zebger I, Wisitruangsakul N, Saggu M, Strack A, Lenz O, Hildebrandt P, Friedrich B (2009) J Biol Chem 284:2159–2168CrossRefPubMedGoogle Scholar
  32. 32.
    Chan Chung KC, Zamble DB (2011) J Biol Chem 286:43081–43090CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Vincent KA, Parkin A, Armstrong FA (2007) Chem Rev 107:4366–4413CrossRefPubMedGoogle Scholar
  34. 34.
    Hamilton CM, Aldea M, Washburn BK, Babitzke P, Kushner SR (1989) J Bacteriol 171:4617–4622PubMedPubMedCentralGoogle Scholar
  35. 35.
    Ize B, Stanley NR, Buchanan G, Palmer T (2003) Mol Microbiol 48:1183–1193CrossRefPubMedGoogle Scholar
  36. 36.
    Pinske C, Krüger S, Soboh B, Ihling C, Kuhns M, Braussemann M, Jaroschinsky M, Sauer C, Sargent F, Sinz A, Sawers RG (2011) Arch Microbiol 193:893–903CrossRefPubMedGoogle Scholar
  37. 37.
    Ballantine SP, Boxer DH (1985) J Bacteriol 163:454–459PubMedPubMedCentralGoogle Scholar
  38. 38.
    Flanagan Lindsey A, Parkin A (2016) Biochem Soc Trans 44:315–328CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Bard AJ, Faulkner LR (2001) Electrochemical methods: fundamentals and applications, 2nd edn. Wiley, New YorkGoogle Scholar
  40. 40.
    Dubini A, Pye RL, Jack RL, Palmer T, Sargent F (2002) Int J Hydrogen Energy 27:1413–1420CrossRefGoogle Scholar
  41. 41.
    Parish D, Benach J, Liu G, Singarapu K, Xiao R, Acton T, Su M, Bansal S, Prestegard J, Hunt J, Montelione G, Szyperski T (2008) J Struct Funct Genomics 9:41–49CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© SBIC 2016

Authors and Affiliations

  • Lisa Bowman
    • 1
  • Jonathan Balbach
    • 2
  • Julia Walton
    • 2
  • Frank Sargent
    • 1
  • Alison Parkin
    • 2
  1. 1.Division of Molecular Microbiology, School of Life SciencesUniversity of DundeeDundeeScotland, UK
  2. 2.Department of ChemistryUniversity of YorkYorkEngland, UK

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