Abstract
Possessing both peroxidase and peroxygenase activities with a broad substrate profile that includes phenols, indoles, and pyrroles, the enzyme dehaloperoxidase (DHP) from Amphitrite ornata is a multifunctional catalytic hemoglobin that challenges many of the assumptions behind the well-established structure–function paradigm in hemoproteins. While previous studies have demonstrated that the F21W variant leads to attenuated peroxidase activity in DHP, here we have studied the impact of this mutation on peroxygenase activity to determine if it is possible to selectively tune DHP to favor one function over another. Biochemical assays with DHP B (F21W) revealed minimal decreases in peroxygenase activity of 1.2–2.1-fold as measured by 4-nitrophenol or 5-Br-indole substrate conversion, whereas the peroxidase activity catalytic efficiency for 2,4,6-trichlorophenol (TCP) was more than sevenfold decreased. Binding studies showed a 20-fold weaker affinity for 5-bromoindole (K d = 2960 ± 940 μM) in DHP B (F21W) compared to WT DHP B. Stopped-flow UV/visible studies and isotope labeling experiments together suggest that the F21W mutation neither significantly changes the nature of the catalytic intermediates, nor alters the mechanisms that have been established for peroxidase and peroxygenase activities in DHP. The X-ray crystal structure (1.96 Å; PDB 5VLX) of DHP B (F21W) revealed that the tryptophan blocks one of the two identified TCP binding sites, specifically TCPinterior, suggesting that the other site, TCPexterior, remains viable for binding peroxygenase substrates. Taken together, these studies demonstrate that blocking the TCPinterior binding site in DHP selectively favors peroxygenase activity at the expense of its peroxidase activity.
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References
Lebioda L, LaCount MW, Zhang E, Chen YP, Han K, Whitton MM, Lincoln DE, Woodin SA (1999) Nature 401:445
Franzen S, Ghiladi RA, Lebioda L, Dawson J (2016) Heme peroxidases. The Royal Society of Chemistry, Cambridge, pp 218–244
Chen YP, Woodin SA, Lincoln DE, Lovell CR (1996) J Biol Chem 271:4609–4612
McCombs NL, D’Antonio J, Barrios DA, Carey LM, Ghiladi RA (2016) Biochemistry 55:2465–2478
Barrios DA, D’Antonio J, McCombs NL, Zhao J, Franzen S, Schmidt AC, Sombers LA, Ghiladi RA (2014) J Am Chem Soc 136:7914–7925
McCombs NL, Smirnova T, Ghiladi RA (2017) Catal Sci Technol 7:3104–3118
Osborne RL, Taylor LO, Han KP, Ely B, Dawson JH (2004) Biochem Biophys Res Commun 324:1194–1198
Feducia J, Dumarieh R, Gilvey LB, Smirnova T, Franzen S, Ghiladi RA (2009) Biochemistry 48:995–1005
D’Antonio J, D’Antonio EL, Thompson MK, Bowden EF, Franzen S, Smirnova T, Ghiladi RA (2010) Biochemistry 49:6600–6616
Thompson MK, Franzen S, Ghiladi RA, Reeder BJ, Svistunenko DA (2010) J Am Chem Soc 132:17501–17510
D’Antonio EL, D’Antonio J, de Serrano V, Gracz H, Thompson MK, Ghiladi RA, Bowden EF, Franzen S (2011) Biochemistry 50:9664–9680
D’Antonio J, Ghiladi RA (2011) Biochemistry 50:5999–6011
Dumarieh R, D’Antonio J, Deliz-Liang A, Smirnova T, Svistunenko DA, Ghiladi RA (2013) J Biol Chem 288:33470–33482
Osborne RL, Sumithran S, Coggins MK, Chen YP, Lincoln DE, Dawson JH (2006) J Inorg Biochem 100:1100–1108
Osborne RL, Coggins MK, Raner GM, Walla M, Dawson JH (2009) Biochemistry 48:4231–4238
Davydov R, Osborne RL, Shanmugam M, Du J, Dawson JH, Hoffman BM (2010) J Am Chem Soc 132:14995–15004
Du J, Sono M, Dawson JH (2010) Biochemistry 49:6064–6069
de Serrano V, D’Antonio J, Franzen S, Ghiladi RA (2010) Acta Cryst D66:529–538
Poulos TL, Kraut J (1980) J Biol Chem 255:8199–8205
Dunford HB (2016) Heme peroxidases. The Royal Society of Chemistry, Cambridge, pp 99–112
Ortiz de Montellano PR (ed) (2015) Cytochrome p450: structure, mechanism, and biochemistry. Springer International Publishing, Switzerland
Franzen S, Thompson MK, Ghiladi RA (2012) Biochim Biophys Acta 1824:578–588
McCombs NL, Moreno-Chicano T, Carey LM, Franzen S, Hough MA, Ghiladi RA (2017) Biochemistry 56:2294–2303
Wang C, Lovelace LL, Sun S, Dawson JH, Lebioda L (2013) Biochemistry 52:6203–6210
LaCount MW, Zhang E, Chen YP, Han K, Whitton MM, Lincoln DE, Woodin SA, Lebioda L (2000) J Biol Chem 275:18712–18716
Thompson MK, Davis MF, de Serrano V, Nicoletti FP, Howes BD, Smulevich G, Franzen S (2010) Biophys J 99:1586–1595
Zhao J, de Serrano V, Le P, Franzen S (2013) Biochemistry 52:2427–2439
Beers RF Jr, Sizer IW (1952) J Biol Chem 195:133–140
Otwinowski Z, Minor W (1997) In: Carter CW Jr, Sweet RM (eds) Methods in enzymology. Academic, New York, pp 307–326
McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) J Appl Crystallogr 40:658–674
Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH (2010) Acta Cryst D66:213–221
Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Acta Cryst D66:486–501
Afonine PV, Grosse-Kunstleve RW, Echols N, Headd JJ, Moriarty NW, Mustyakimov M, Terwilliger TC, Urzhumtsev A, Zwart PH, Adams PD (2012) Acta Cryst D68:352–367
Chenprakhon P, Sucharitakul J, Panijpan B, Chaiyen P (2010) J Chem Educ 87:829–831
Carugo O, Pongor S (2001) Protein Sci 10:1470–1473
Cohen FE, Sternberg MJ (1980) J Mol Biol 138:321–333
Chothia C, Lesk AM (1986) EMBO J 5:823–826
Sun S, Sono M, Wang C, Du J, Lebioda L, Dawson JH (2014) Arch Biochem Biophys 545:108–115
Jiang S, Wright I, Swartz P, Franzen S (2013) Biochimica et Biophysica Acta (BBA) Proteins and Proteomics 1834:2020–2029
Zhao J, de Serrano V, Dumarieh R, Thompson M, Ghiladi RA, Franzen S (2012) J Phys Chem B 116:12065–12077
Carey LM, Gavenko R, Svistunenko DA, Ghiladi RA (2017) Biochimica et Biophysica Acta (BBA) Proteins and Proteomics (in press)
de Serrano VS, Davis MF, Gaff JF, Zhang Q, Chen Z, D’Antonio EL, Bowden EF, Rose R, Franzen S (2010) Acta Cryst D66:770–782
Chen Z, de Serrano V, Betts L, Franzen S (2009) Acta Cryst D65:34–40
de Serrano V, Chen Z, Davis MF, Franzen S (2007) Acta Cryst D63:1094–1101
Kwon H, Moody PCE, Raven EL (2016) Heme peroxidases. The Royal Society of Chemistry, Cambridge, pp 47–60
Acknowledgements
This project was supported by NSF CAREER Award CHE-1150709 and NSF CHE-1609446. Mass spectra were obtained at the Mass Spectrometry Facility for Biotechnology at North Carolina State University. Partial funding for the facility was obtained from the North Carolina Biotechnology Center and the National Science Foundation.
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Carey, L.M., Kim, K.B., McCombs, N.L. et al. Selective tuning of activity in a multifunctional enzyme as revealed in the F21W mutant of dehaloperoxidase B from Amphitrite ornata . J Biol Inorg Chem 23, 209–219 (2018). https://doi.org/10.1007/s00775-017-1520-x
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DOI: https://doi.org/10.1007/s00775-017-1520-x