BlsE, a predicted radical S-adenosyl-L-methionine (SAM) protein, was anaerobically purified and reconstituted in vitro to study its function in the blasticidin S biosynthetic pathway. The putative role of BlsE was elucidated based on bioinformatics analysis, genetic inactivation and biochemical characterization. Biochemical results showed that BlsE is a SAM-dependent radical enzyme that utilizes cytosylglucuronic acid, the accumulated intermediate metabolite in blsE mutant, as substrate and catalyzes decarboxylation at the C5 position of the glucoside residue to yield cytosylarabinopyranose. Additionally, we report the purification and reconstitution of BlsE, characterization of its [4Fe–4S] cluster using UV-vis and electron paramagnetic resonance (EPR) spectroscopic analysis, and investigation of the ability of flavodoxin (Fld), flavodoxin reductase (Fpr) and NADPH to reduce the [4Fe–4S]2+ cluster. Mutagenesis studies demonstrated that Cys31, Cys35, Cys38 in the C×××C×MC motif and Gly73, Gly74, Glu75, Pro76 in the GGEP motif were crucial amino acids for BlsE activity while mutation of Met37 had little effect on its function. Our results indicate that BlsE represents a typical [4Fe–4S]-containing radical SAM enzyme and it catalyzes decarboxylation in blasticidin S biosynthesis.
References
[1]
Huang KT, Misato T, Asuyama H (1964) Effect of Blasticidin S on Protein Synthesis of Piricularia Oryzae. J Antibiot (Tokyo) 17: 65–70.
[2]
Yamaguchi H, Yamamoto C, Tanaka N (1965) Inhibition of protein synthesis by blasticidin S. I. Studies with cell-free systems from bacterial and mammalian cells. J Biochem 57: 667–677.
[3]
Tamura K, Kimura M, Yamaguchi I (1995) Blasticidin S deaminase gene (BSD): a new selection marker gene for transformation of Arabidopsis thaliana and Nicotiana tabacum. Biosci Biotechnol Biochem 59: 2336–2338.
[4]
Kamakura T, Yoneyama K, Yamaguchi I (1990) Expression of the blasticidin S deaminase gene (bsr) in tobacco: fungicide tolerance and a new selective marker for transgenic plants. Mol Gen Genet 223: 332–334.
[5]
Mamoun CB, Gluzman IY, Goyard S, Beverley SM, Goldberg DE (1999) A set of independent selectable markers for transfection of the human malaria parasite Plasmodium falciparum. Proc Natl Acad Sci U S A 96: 8716–8720.
[6]
Kimura M, Takatsuki A, Yamaguchi I (1994) Blasticidin S deaminase gene from Aspergillus terreus (BSD): a new drug resistance gene for transfection of mammalian cells. Biochim Biophys Acta 1219: 653–659.
[7]
Cone MC, Yin X, Grochowski LL, Parker MR, Zabriskie TM (2003) The blasticidin S biosynthesis gene cluster from Streptomyces griseochromogenes: sequence analysis, organization, and initial characterization. ChemBiochem 4: 821–828.
[8]
Wu J, Li L, Deng Z, Zabriskie TM, He X (2012) Analysis of the mildiomycin biosynthesis gene cluster in Streptoverticillum remofaciens ZJU5119 and characterization of MilC, a hydroxymethyl cytosyl-glucuronic acid synthase. ChemBiochem 13: 1613–1621.
[9]
Li L, Xu Z, Xu X, Wu J, Zhang Y, et al. (2008) The mildiomycin biosynthesis: initial steps for sequential generation of 5-hydroxymethylcytidine 5'-monophosphate and 5-hydroxymethylcytosine in Streptoverticillium rimofaciens ZJU5119. ChemBiochem 9: 1286–1294.
[10]
Frey PA, Hegeman AD, Ruzicka FJ (2008) The Radical SAM Superfamily. Crit Rev Biochem Mol Biol 43: 63–88.
[11]
Sofia HJ, Chen G, Hetzler BG, Reyes-Spindola JF, Miller NE (2001) Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods. Nucleic Acids Res 29: 1097–1106.
[12]
Frey PA, Booker SJ (2001) Radical mechanisms of S-adenosylmethionine-dependent enzymes. Adv Protein Chem 58: 1–45.
[13]
Haldar S, Paul S, Joshi N, Dasgupta A, Chattopadhyay K (2012) The presence of the iron-sulfur motif is important for the conformational stability of the antiviral protein, Viperin. PLoS One 7: e31797.
[14]
Lotierzo M, Tse Sum Bui B, Florentin D, Escalettes F, Marquet A (2005) Biotin synthase mechanism: an overview. Biochem Soc Trans 33: 820–823.
[15]
Shaw NM, Birch OM, Tinschert A, Venetz V, Dietrich R, et al. (1998) Biotin synthase from Escherichia coli: isolation of an enzyme-generated intermediate and stoichiometry of S-adenosylmethionine use. Biochem J 330 (Pt 3): 1079–1085.
[16]
Cicchillo RM, Booker SJ (2005) Mechanistic investigations of lipoic acid biosynthesis in Escherichia coli: both sulfur atoms in lipoic acid are contributed by the same lipoyl synthase polypeptide. J AM Chem Soc 127: 2860–2861.
[17]
Hernandez HL, Pierrel F, Elleingand E, Garcia-Serres R, Huynh BH, et al. (2007) MiaB, a bifunctional radical-S-adenosylmethionine enzyme involved in the thiolation and methylation of tRNA, contains two essential [4Fe–4S] clusters. Biochemistry 46: 5140–5147.
[18]
Wecksler SR, Stoll S, Iavarone AT, Imsand EM, Tran H, et al. (2010) Interaction of PqqE and PqqD in the pyrroloquinoline quinone (PQQ) biosynthetic pathway links PqqD to the radical SAM superfamily. Chem Commun (Camb) 46: 7031–7033.
[19]
Wecksler SR, Stoll S, Tran H, Magnusson OT, Wu SP, et al. (2009) Pyrroloquinoline quinone biogenesis: demonstration that PqqE from Klebsiella pneumoniae is a radical S-adenosyl-L-methionine enzyme. Biochemistry 48: 10151–10161.
[20]
Chatterjee A, Li Y, Zhang Y, Grove TL, Lee M, et al. (2008) Reconstitution of ThiC in thiamine pyrimidine biosynthesis expands the radical SAM superfamily. Nat Chem Biol 4: 758–765.
[21]
Zhang Q, Li Y, Chen D, Yu Y, Duan L, et al. (2011) Radical-mediated enzymatic carbon chain fragmentation-recombination. Nat Chem Biol 7: 154–160.
[22]
Nicolet Y, Amara P, Mouesca JM, Fontecilla-Camps JC (2009) Unexpected electron transfer mechanism upon AdoMet cleavage in radical SAM proteins. Proc Natl Acad Sci U S A 106: 14867–14871.
[23]
Dowling DP, Vey JL, Croft AK, Drennan CL (2012) Structural diversity in the AdoMet radical enzyme superfamily. Biochim Biophys Acta 1824: 1178–1195.
[24]
Yokoyama K, Numakura M, Kudo F, Ohmori D, Eguchi T (2007) Characterization and mechanistic study of a radical SAM dehydrogenase in the biosynthesis of butirosin. J Am Chem Soc 129: 15147–15155.
[25]
Chatterjee A, Hazra AB, Abdelwahed S, Hilmey DG, Begley TP (2010) A “radical dance” in thiamin biosynthesis: mechanistic analysis of the bacterial hydroxymethylpyrimidine phosphate synthase. Angew Chem Int Ed Engl 49: 8653–8656.
[26]
Wagner AF, Frey M, Neugebauer FA, Schafer W, Knappe J (1992) The free radical in pyruvate formate-lyase is located on glycine-734. Proc Natl Acad Sci U S A 89: 996–1000.
[27]
Krebs C, Broderick WE, Henshaw TF, Broderick JB, Huynh BH (2002) Coordination of adenosylmethionine to a unique iron site of the [4Fe-4S] of pyruvate formate-lyase activating enzyme: a Mossbauer spectroscopic study. J AM Chem Soc 124: 912–913.
[28]
Torrents E, Buist G, Liu A, Eliasson R, Kok J, et al. (2000) The anaerobic (class III) ribonucleotide reductase from Lactococcus lactis. Catalytic properties and allosteric regulation of the pure enzyme system. J Biol Chem 275: 2463–2471.
[29]
Szu PH, Ruszczycky MW, Choi SH, Yan F, Liu HW (2009) Characterization and mechanistic studies of DesII: a radical S-adenosyl-L-methionine enzyme involved in the biosynthesis of TDP-D-desosamine. J AM Chem Soc 131: 14030–14042.
[30]
Frey PA, Magnusson OT (2003) S-Adenosylmethionine: a wolf in sheep's clothing, or a rich man's adenosylcobalamin? Chem Rev 103: 2129–2148.
[31]
Gambarelli S, Mulliez E, Fontecave M (2010) Iron–Sulfur Clusters in “Radical SAM” Enzymes: Spectroscopy and Coordination. Metals in Biology 29: 53–82.
[32]
Vey JL, Drennan CL (2011) Structural insights into radical generation by the radical SAM superfamily. Chem Rev 111: 2487–2506.
[33]
Berkovitch F, Nicolet Y, Wan JT, Jarrett JT, Drennan CL (2004) Crystal structure of biotin synthase, an S-adenosylmethionine-dependent radical enzyme. Science 303: 76–79.
[34]
Grove TL, Ahlum JH, Sharma P, Krebs C, Booker SJ (2010) A consensus mechanism for Radical SAM-dependent dehydrogenation? BtrN contains two [4Fe-4S] clusters. Biochemistry 49: 3783–3785.
[35]
Liu A, Graslund A (2000) Electron paramagnetic resonance evidence for a novel interconversion of [3Fe–4S](+) and [4Fe–4S](+) clusters with endogenous iron and sulfide in anaerobic ribonucleotide reductase activase in vitro. J Biol Chem 275: 12367–12373.
[36]
Zhang Q, Chen D, Lin J, Liao R, Tong W, et al. (2011) Characterization of NocL involved in thiopeptide nocathiacin I biosynthesis: a [4Fe-4S] cluster and the catalysis of a radical S-adenosylmethionine enzyme. J Biol Chem 286: 21287–21294.
[37]
Sancho J (2006) Flavodoxins: sequence, folding, binding, function and beyond. Cell Mol Life Sci 63: 855–864.
[38]
Sanyal I, Cohen G, Flint DH (1994) Biotin synthase: purification, characterization as a [2Fe-2S]cluster protein, and in vitro activity of the Escherichia coli bioB gene product. Biochemistry 33: 3625–3631.
[39]
Nicolet Y, Drennan CL (2004) AdoMet radical proteins–from structure to evolution–alignment of divergent protein sequences reveals strong secondary structure element conservation. Nucleic Acids Res 32: 4015–4025.
[40]
Layer G, Moser J, Heinz DW, Jahn D, Schubert WD (2003) Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of Radical SAM enzymes. EMBO J 22: 6214–6224.
[41]
Bali S, Lawrence AD, Lobo SA, Saraiva LM, Golding BT, et al. (2011) Molecular hijacking of siroheme for the synthesis of heme and d1 heme. Proc Natl Acad Sci U S A 108: 18260–18265.
[42]
Lobo SA, Brindley A, Warren MJ, Saraiva LM (2009) Functional characterization of the early steps of tetrapyrrole biosynthesis and modification in Desulfovibrio vulgaris Hildenborough. Biochem J 420: 317–325.
[43]
Kennedy MC, Kent TA, Emptage M, Merkle H, Beinert H, et al. (1984) Evidence for the formation of a linear [3Fe–4S] cluster in partially unfolded aconitase. J Biol Chem 259: 14463–14471.
[44]
Beinert H (1983) Semi-micro methods for analysis of labile sulfide and of labile sulfide plus sulfane sulfur in unusually stable iron-sulfur proteins. Anal Biochem 131: 373–378.
[45]
Layer G, Grage K, Teschner T, Schunemann V, Breckau D, et al. (2005) Radical S-adenosylmethionine enzyme coproporphyrinogen III oxidase HemN: functional features of the [4Fe–4S] cluster and the two bound S-adenosyl-L-methionines. J Biol Chem 280: 29038–29046.
