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Solution structure and biochemical characterization of a spare part protein that restores activity to an oxygen-damaged glycyl radical enzyme

  • Sarah E. J. Bowman
  • Lindsey R. F. Backman
  • Rebekah E. Bjork
  • Mary C. Andorfer
  • Santiago Yori
  • Alessio Caruso
  • Collin M. Stultz
  • Catherine L. DrennanEmail author
Original Paper
Part of the following topical collections:
  1. Joan Broderick: Papers in Celebration of Her 2019 ACS Alfred Bader Award in Bioinorganic or Bioorganic Chemistry

Abstract

Glycyl radical enzymes (GREs) utilize a glycyl radical cofactor to carry out a diverse array of chemically challenging enzymatic reactions in anaerobic bacteria. Although the glycyl radical is a powerful catalyst, it is also oxygen sensitive such that oxygen exposure causes cleavage of the GRE at the site of the radical. This oxygen sensitivity presents a challenge to facultative anaerobes dwelling in areas prone to oxygen exposure. Once GREs are irreversibly oxygen damaged, cells either need to make new GREs or somehow repair the damaged one. One particular GRE, pyruvate formate lyase (PFL), can be repaired through the binding of a 14.3 kDa protein, termed YfiD, which is constitutively expressed in E. coli. Herein, we have solved a solution structure of this ‘spare part’ protein using nuclear magnetic resonance spectroscopy. These data, coupled with data from circular dichroism, indicate that YfiD has an inherently flexible N-terminal region (residues 1–60) that is followed by a C-terminal region (residues 72–127) that has high similarity to the glycyl radical domain of PFL. Reconstitution of PFL activity requires that YfiD binds within the core of the PFL barrel fold; however, modeling suggests that oxygen-damaged, i.e. cleaved, PFL cannot fully accommodate YfiD. We further report that a PFL variant that mimics the oxygen-damaged enzyme is highly susceptible to proteolysis, yielding additionally truncated forms of PFL. One such PFL variant of ~ 77 kDa makes an ideal scaffold for the accommodation of YfiD. A molecular model for the rescue of PFL activity by YfiD is presented.

Graphic abstract

Keywords

Radical chemistry Glycyl radical enzyme Nuclear magnetic resonance Cofactor repair Circular dichroism 

Abbreviations

5′-dAdo

5′-deoxyadenosyl

AdoCbl

Adenosylcobalamin

AdoMet

S-adenosylmethionine

BSS

Benzylsuccinate synthase

CD

Circular dichroism

cPFL

Cleaved pyruvate formate lyase

GDH

Glycerol/propanediol dehydratase

GrcA

Autonomous glycyl radical cofactor

GRD

Glycyl radical domain

GRE

Glycyl radical enzyme

HSQC

Heteronuclear single quantum coherence

IMAC

Immobilized metal affinity chromatography

NMR

Nuclear magnetic resonance

NOE

Nuclear Overhauser effect

PFL

Pyruvate formate lyase

PFL-AE

Pyruvate formate lyase activating enzyme

RNR

Ribonucleotide reductase

t1PFL

PFL truncation product 1 at 77 kDa

t2PFL

PFL truncation product 2 at 71 kDa

truncYfiD

Truncated YfiD with 60 N-terminal residues removed

Notes

Acknowledgements

This work was supported in part by the National Institutes of Health (NIH) GM069857 (C.L.D.), R35 GM126982 (C.L.D.), F32 GM129882 (M.C.A.), and F32 GM099257 (S.E.J.B.), Analog Devices (C.M.S.), MIT-IBM Watson Lab (C.M.S.), MIT J-Clinic (C.M.S.), and the National Science Foundation (NSF) Graduate Research Fellowship under Grant No. 1122374 (L.R.F.B.). C.L.D is a Howard Hughes Medical Institute (HHMI) Investigator. S.Y. and L.R.F.B. were part of the HHMI EXROP program and the Bio-MIT Summer Research Program (MSRP) program. A.C. was also part of the MSRP program. L.R.F.B. is a recipient of a Dow Fellowship at MIT and a Gilliam Fellowship from HHMI. The Biophysical Instrumentation Facility for the Study of Complex Macromolecular Systems (NSF-0070319) is gratefully acknowledged. We would like to thank University of Connecticut Health Center NMR facility and Massachusetts Institute of Technology’s Francis Bitter Magnet Lab for instrument use. We acknowledge Miranda Lynch for helping with R scripting, and Laurel Kinman for helping with editing.

Author Contributions

SEJB and CLD designed research; SEJB, LRFB, REB, SY, MCA, and AC performed experiments; SEJB, LRFB, SY, CMS, and CLD analyzed data; and the manuscript was written by SEJB, LRFB, CLD, and MCA

Supplementary material

Supplementary material 1 (MP4 65801 kb)

775_2019_1681_MOESM2_ESM.pdf (1.3 mb)
Supplementary material 2 (PDF 1370 kb)

References

  1. 1.
    Backman LRF, Funk MA, Dawson CD, Drennan CL (2017) New tricks for the glycyl radical enzyme family. Crit Rev Biochem Mol Biol 52:674–695CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Broderick JB, Duffus BR, Duschene KS, Shepard EM (2014) Radical S-adenosylmethionine enzymes. Chem Rev 114:4229–4317CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Banerjee R (2003) Radical carbon skeleton rearrangements: catalysis by coenzyme B12-dependent mutases. Chem Rev 103:2083–2094CrossRefPubMedGoogle Scholar
  4. 4.
    Hausinger RP (2004) FeII/alpha-ketoglutarate-dependent hydroxylases and related enzymes. Crit Rev Biochem Mol Biol 39:21–68CrossRefPubMedGoogle Scholar
  5. 5.
    Stubbe J, van Der Donk WA (1998) Protein radicals in enzyme catalysis. Chem Rev 98:705–762CrossRefPubMedGoogle Scholar
  6. 6.
    Frey PA (1990) Importance of organic radicals in enzymatic cleavage of unactivated C–H bonds. Chem Rev 90:1343–1357CrossRefGoogle Scholar
  7. 7.
    Warren MJ, Raux E, Schubert HL, Escalante-Semerena JC (2002) The biosynthesis of adenosylcobalamin (vitamin B12). Nat Prod Rep 19:390–412CrossRefPubMedGoogle Scholar
  8. 8.
    Scott AI, Roessner CA (2002) Biosynthesis of cobalamin (vitamin B(12)). Biochem Soc Trans 30:613–620CrossRefPubMedGoogle Scholar
  9. 9.
    Conradt H, Hohmann-Berger M, Hohmann HP, Blaschkowski HP, Knappe J (1984) Pyruvate formate-lyase (inactive form) and pyruvate formate-lyase activating enzyme of Escherichia coli: isolation and structural properties. Arch Biochem Biophys 228:133–142CrossRefPubMedGoogle Scholar
  10. 10.
    Henshaw TF, Cheek J, Broderick JB (2000) The [4Fe–4S]1+ cluster of pyruvate formate-lyase activating enzyme generates the glycyl radical on pyruvate formate-lyase: EPR-detected single turnover. J Am Chem Soc 122:8331–8332CrossRefGoogle Scholar
  11. 11.
    Frey PA (1993) Lysine 2,3-aminomutase: is adenosylmethionine a poor man’s adenosylcobalamin? FASEB J 7:662–670CrossRefPubMedGoogle Scholar
  12. 12.
    Bridwell-Rabb J, Grell TAJ, Drennan CL (2018) A rich man, poor man story of S-adenosylmethionine and cobalamin revisited. Annu Rev Biochem 87:555–584CrossRefPubMedGoogle Scholar
  13. 13.
    Frey PA, Ballinger MD, Reed GH (1998) S-adenosylmethionine: a ‘poor man’s coenzyme B12’ in the reaction of lysine 2,3-aminomutase. Biochem Soc Trans 26:304–310CrossRefPubMedGoogle Scholar
  14. 14.
    Knappe J, Blaschkowski HP, Grobner P, Schmitt T (1974) Pyruvate formate-lyase of Escherichia coli: the acetyl-enzyme intermediate. Eur J Biochem 50:253–263CrossRefPubMedGoogle Scholar
  15. 15.
    Knappe J, Wagner AF (1995) Glycyl free radical in pyruvate formate-lyase: synthesis, structure characteristics, and involvement in catalysis. Methods Enzymol 258:343–362CrossRefPubMedGoogle Scholar
  16. 16.
    Knappe J, Sawers G (1990) A radical-chemical route to acetyl-CoA: the anaerobically induced pyruvate formate-lyase system of Escherichia coli. FEMS Microbiol Rev 6:383–398PubMedGoogle Scholar
  17. 17.
    Sawers G, Suppmann B (1992) Anaerobic induction of pyruvate formate-lyase gene expression is mediated by the ArcA and FNR proteins. J Bacteriol 174:3474–3478CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Partridge JD, Sanguinetti G, Dibden DP, Roberts RE, Poole RK, Green J (2007) Transition of Escherichia coli from aerobic to micro-aerobic conditions involves fast and slow reacting regulatory components. J Biol Chem 282:11230–11237CrossRefPubMedGoogle Scholar
  19. 19.
    Han MJ, Yoon SS, Lee SY (2001) Proteome analysis of metabolically engineered Escherichia coli producing Poly(3-hydroxybutyrate). J Bacteriol 183:301–308CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Wagner AF, Schultz S, Bomke J, Pils T, Lehmann WD, Knappe J (2001) YfiD of Escherichia coli and Y06I of bacteriophage T4 as autonomous glycyl radical cofactors reconstituting the catalytic center of oxygen-fragmented pyruvate formate-lyase. Biochem Biophys Res Commun 285:456–462CrossRefPubMedGoogle Scholar
  21. 21.
    Marshall FA, Messenger SL, Wyborn NR, Guest JR, Wing H, Busby SJ, Green J (2001) A novel promoter architecture for microaerobic activation by the anaerobic transcription factor FNR. Mol Microbiol 39:747–753CrossRefPubMedGoogle Scholar
  22. 22.
    Wyborn NR, Messenger SL, Henderson RA, Sawers G, Roberts RE, Attwood MM, Green J (2002) Expression of the Escherichia coli yfiD gene responds to intracellular pH and reduces the accumulation of acidic metabolic end products. Microbiology 148:1015–1026CrossRefPubMedGoogle Scholar
  23. 23.
    Kumar R, Shimizu K (2011) Transcriptional regulation of main metabolic pathways of cyoA, cydB, fnr, and fur gene knockout Escherichia coli in C-limited and N-limited aerobic continuous cultures. Microb Cell Fact 10:3CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Green J, Baldwin ML (1997) HlyX, the FNR homologue of Actinobacillus pleuropneumoniae, is a [4Fe–4S]-containing oxygen-responsive transcription regulator that anaerobically activates FNR-dependent class I promoters via an enhanced AR1 contact. Mol Microbiol 24:593–605CrossRefPubMedGoogle Scholar
  25. 25.
    Vey JL, Yang J, Li M, Broderick WE, Broderick JB, Drennan CL (2008) Structural basis for glycyl radical formation by pyruvate formate-lyase activating enzyme. Proc Natl Acad Sci USA 105:16137–16141CrossRefPubMedGoogle Scholar
  26. 26.
    Peng Y, Veneziano SE, Gillispie GD, Broderick JB (2010) Pyruvate formate-lyase, evidence for an open conformation favored in the presence of its activating enzyme. J Biol Chem 285:27224–27231CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Dunker AK, Lawson JD, Brown CJ, Williams RM, Romero P, Oh JS, Oldfield CJ, Campen AM, Ratliff CM, Hipps KW, Ausio J, Nissen MS, Reeves R, Kang C, Kissinger CR, Bailey RW, Griswold MD, Chiu W, Garner EC, Obradovic Z (2001) Intrinsically disordered protein. J Mol Graph Model 19:26–59CrossRefPubMedGoogle Scholar
  28. 28.
    Kelly SM, Jess TJ, Price NC (2005) How to study proteins by circular dichroism. Biochim Biophys Acta 1751:119–139CrossRefPubMedGoogle Scholar
  29. 29.
    Easton CJ, Hay MP (1986) Preferential reactivity of glycine residues in free radical reactions of amino acid derivatives. J Chem Soc Chem Commun 1:55–57CrossRefGoogle Scholar
  30. 30.
    Funk MA, Judd ET, Marsh EN, Elliott SJ, Drennan CL (2014) Structures of benzylsuccinate synthase elucidate roles of accessory subunits in glycyl radical enzyme activation and activity. Proc Natl Acad Sci USA 111:10161–10166CrossRefPubMedGoogle Scholar
  31. 31.
    Li L, Patterson DP, Fox CC, Lin B, Coschigano PW, Marsh EN (2009) Subunit structure of benzylsuccinate synthase. Biochemistry 48:1284–1292CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Coschigano PW, Bishop BJ (2004) Role of benzylsuccinate in the induction of the tutE tutFDGH gene complex of T. aromatica strain T1. FEMS Microbiol Lett 231:261–266CrossRefPubMedGoogle Scholar
  33. 33.
    O’Brien JR, Raynaud C, Croux C, Girbal L, Soucaille P, Lanzilotta WN (2004) Insight into the mechanism of the B12-independent glycerol dehydratase from Clostridium butyricum: preliminary biochemical and structural characterization. Biochemistry 43:4635–4645CrossRefPubMedGoogle Scholar
  34. 34.
    Sun X, Ollagnier S, Schmidt PP, Atta M, Mulliez E, Lepape L, Eliasson R, Graslund A, Fontecave M, Reichard P, Sjoberg BM (1996) The free radical of the anaerobic ribonucleotide reductase from Escherichia coli is at glycine 681. J Biol Chem 271:6827–6831CrossRefPubMedGoogle Scholar
  35. 35.
    Team, RC. (2017) R: a language and environment for statistical computingGoogle Scholar
  36. 36.
    Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293CrossRefPubMedGoogle Scholar
  37. 37.
    Lee W, Tonelli M, Markley JL (2015) NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics 31:1325–1327CrossRefPubMedGoogle Scholar
  38. 38.
    Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, Llinas M, Ulrich EL, Markley JL, Ionides J, Laue ED (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 59:687–696CrossRefPubMedGoogle Scholar
  39. 39.
    Guntert P (2009) Automated structure determination from NMR spectra. Eur Biophys J 38:129–143CrossRefPubMedGoogle Scholar

Copyright information

© Society for Biological Inorganic Chemistry (SBIC) 2019

Authors and Affiliations

  • Sarah E. J. Bowman
    • 1
    • 2
    • 7
  • Lindsey R. F. Backman
    • 2
    • 4
  • Rebekah E. Bjork
    • 1
  • Mary C. Andorfer
    • 1
    • 3
  • Santiago Yori
    • 4
    • 8
  • Alessio Caruso
    • 4
    • 9
  • Collin M. Stultz
    • 5
    • 10
  • Catherine L. Drennan
    • 1
    • 2
    • 3
    • 6
    Email author
  1. 1.Howard Hughes Medical InstituteMassachusetts Institute of TechnologyCambridgeUSA
  2. 2.Department of ChemistryMassachusetts Institute of TechnologyCambridgeUSA
  3. 3.Department of BiologyMassachusetts Institute of TechnologyCambridgeUSA
  4. 4.MIT Summer Research Program (MSRP)Massachusetts Institute of TechnologyCambridgeUSA
  5. 5.Electrical Engineering and Computer Science and Institute for Medical Engineering and ScienceMassachusetts Institute of TechnologyCambridgeUSA
  6. 6.Center for Environmental HealthMassachusetts Institute of TechnologyCambridgeUSA
  7. 7.Hauptman-Woodward Medical Research InstituteBuffaloUSA
  8. 8.Department of Molecular and Cell BiologyUniversity of California BerkeleyBerkeleyUSA
  9. 9.Department of ChemistryPrinceton UniversityPrincetonUSA
  10. 10.Division of Cardiovascular MedicineMassachusetts General HospitalBostonUSA

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