4-Hydroxyphenylacetate decarboxylase (4Hpad) is the prototype of a new class of Fe-S cluster-dependent glycyl radical enzymes (Fe-S GREs) acting on aromatic compounds. The two-enzyme component system comprises a decarboxylase responsible for substrate conversion and a dedicated activating enzyme (4Hpad-AE). The decarboxylase uses a glycyl/thiyl radical dyad to convert 4-hydroxyphenylacetate into p-cresol (4-methylphenol) by a biologically unprecedented Kolbe-type decarboxylation. In addition to the radical dyad prosthetic group, the decarboxylase unit contains two [4Fe-4S] clusters coordinated by an extra small subunit of unknown function. 4Hpad-AE reductively cleaves S-adenosylmethionine (SAM or AdoMet) at a site-differentiated [4Fe-4S]2+/+ cluster (RS cluster) generating a transient 5′-deoxyadenosyl radical that produces a stable glycyl radical in the decarboxylase by the abstraction of a hydrogen atom. 4Hpad-AE binds up to two auxiliary [4Fe-4S] clusters coordinated by a ferredoxin-like insert that is C-terminal to the RS cluster-binding motif. The ferredoxin-like domain with its two auxiliary clusters is not vital for SAM-dependent glycyl radical formation in the decarboxylase, but facilitates a longer lifetime for the radical. This review describes the 4Hpad and cognate AE families and focuses on the recent advances and open questions concerning the structure, function and mechanism of this novel Fe-S-dependent class of GREs.

1.
Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25:3389-3402.
[PubMed]
2.
Andrei PI, Pierik AJ, Zauner S, Andrei-Selmer LC, Selmer T: Subunit composition of the glycyl radical enzyme p-hydroxyphenylacetate decarboxylase. A small subunit, HpdC, is essential for catalytic activity. Eur J Biochem 2004;271:2225-2230.
[PubMed]
3.
Bammens B, Evenepoel P, Keuleers H, Verbeke K, Vanrenterghem Y: Free serum concentrations of the protein-bound retention solute p-cresol predict mortality in hemodialysis patients. Kidney Int 2006;69:1081-1087.
[PubMed]
4.
Bashford D, Karplus M: Pkas of ionizable groups in proteins - atomic detail from a continuum electrostatic model. Biochemistry 1990;29:10219-10225.
[PubMed]
5.
Becker A, Fritz-Wolf K, Kabsch W, Knappe J, Schultz S, Wagner AFV: Structure and mechanism of the glycyl radical enzyme pyruvate formate-lyase. Nat Struct Biol 1999;6:969-975.
[PubMed]
6.
Bennett BD, Kimball EH, Gao M, Osterhout R, Van Dien SJ, Rabinowitz JD: Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat Chem Biol 2009;5:593-599.
[PubMed]
7.
Bombarda E, Ullmann GM: pH-dependent pKa values in proteins - a theoretical analysis of protonation energies with practical consequences for enzymatic reactions. J Phys Chem B 2010;114:1994-2003.
[PubMed]
8.
Buckel W: Radical and electron recycling in catalysis. Angew Chem Int Ed Engl 2009;48:6779-6787.
[PubMed]
9.
Buckel W, Golding BT: Radical species in the catalytic pathways of enzymes from anaerobes. FEMS Microbiol Rev 1999;22:523-541.
10.
D'Ari L, Barker HA: p-Cresol formation by cell-free extracts of Clostridium difficile. Arch Microbiol 1985;143:311-312.
[PubMed]
11.
Dawson LF, Stabler RA, Wren BW: Assessing the role of p-cresol tolerance in Clostridia difficile. J Med Microbiol 2008;57:745-749.
[PubMed]
12.
Demick JM, Lanzilotta WN: Radical SAM activation of the B12-independent glycerol dehydratase results in formation of 5′-deoxy-5′-(methylthio)adenosine and not 5′-deoxyadenosine. Biochemistry 2011;50:440-442.
[PubMed]
13.
Dey A, Peng Y, Broderick WE, Hedman B, Hodgson KO, Broderick JB, Solomon EI: S K-edge XAS and DFT calculations on SAM dependent pyruvate formate-lyase activating enzyme: nature of interaction between the Fe4S4 cluster and SAM and its role in reactivity. J Am Chem Soc 2011;133:18656-18662.
[PubMed]
14.
Eklund H, Fontecave M: Glycyl radical enzymes: a conservative structural basis for radicals. Structure 1999;7:257-262.
[PubMed]
15.
Elsden SR, Hilton MG, Waller JM: End products of metabolism of aromatic amino acids by Clostridia. Arch Microbiol 1976;107:283-288.
[PubMed]
16.
Feliks M, Martins BM,Ullmann GM: Catalytic mechanism of the glycyl radical enzyme 4-hydroxyphenylacetate decarboxylase from continuum electrostatic and QC/MM calculations. J Am Chem Soc 2013;135:14574-14585.
[PubMed]
17.
Feliks M, Ullmann GM: Glycerol dehydratation by the B12-independent enzyme may not involve the migration of a hydroxyl group: a computational study. J Phys Chem B 2012;116:7076-7087.
[PubMed]
18.
Frey PA: Radical mechanisms of enzymatic catalysis. Annu Rev Biochem 2001;70:121-148.
[PubMed]
19.
Frey PA, Hegeman AD, Reed GH: Free radical mechanisms in enzymology. Chem Rev 2006;106:3302-3316.
[PubMed]
20.
Frey PA, Hegeman AD, Ruzicka FJ: The radical SAM superfamily. Crit Rev Biochem Mol Biol 2008;43:63-88.
[PubMed]
21.
Funk MA, Judd ET, Marsh ENG, Elliott SJ, Drennan, CL: Structures of benzylsuccinate synthase elucidate roles of accessory subunits in glycyl radical enzyme activation and activity. Proc Natl Acad Sci USA 2014;111:10161-10166.
[PubMed]
22.
Gambarelli S, Luttringer F, Padovani D, Mulliez E, Fontecave M: Activation of the anaerobic ribonucleotide reductase by S-adenosylmethionine. Chem Biochem 2005;6:1960-1962.
[PubMed]
23.
Goldman PJ, Grove TL, Sites LA, McLaughlin MI, Booker SJ, Drennan, CL: X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification. Proc Natl Acad Sci USA 2013;110:8519-8524.
[PubMed]
24.
Hafiz S, Oakley CL: Clostridium difficile - isolation and characteristics. J Med Microbiol 1976;9:129-136.
[PubMed]
25.
Hioe, J, Savasci G Brand H, Zipse H: The stability of Cα peptide radicals: why glycyl radical enzymes? Chemistry 2011;17:3781-3789.
[PubMed]
26.
Knappe J, Neugebauer FA, Blaschkowski HP, Gänzler M: Post-translational activation introduces a free radical into pyruvate formate-lyase. Proc Natl Acad Sci USA 1984;81:1332-1335.
[PubMed]
27.
Komuro M, Higuchi T, Hirobe M: Application of chemical cytochrome P-450 model systems to studies on drug metabolism-VIII. Novel metabolism of carboxylic acids via oxidative decarboxylation. Bioorg Med Chem 1995;3:55-65.
[PubMed]
28.
Kuchenreuther JM, Myers WK, Stich TA, George SJ, Nejatyjahromy Y, Swartz JR, Britt RD: A radical intermediate in tyrosine scission to the CO and CN- ligands of Fe-Fe hydrogenase. Science 2013;342:472-475.
[PubMed]
29.
Lanz ND, Booker SJ: Identification and function of auxiliary iron-sulfur clusters in radical SAM enzymes. Biochim Biophys Acta 2012;1824:1196-1212.
[PubMed]
30.
Lehtiö L, Grossmann JG, Kokona B, Fairman R, Goldman A: Crystal structure of a glycyl radical enzyme from Archaeoglobus fulgidus. J Mol Biol 2006;357:221-235.
[PubMed]
31.
Leuthner B, Leutwein C, Schulz H, Horth P, Haehnel W, Schiltz E, Schagger H, Heider J: Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism. Mol Microbiol 1998;28:615-628.
[PubMed]
32.
Li L, Patterson DP, Fox CC, Lin B, Coshigano PW, Marsh ENG: Subunit structure of benzylsuccinate synthase. Biochemistry 2009;48:1284-1292.
[PubMed]
33.
Logan DT, Andersson J, Sjoberg BM, Nordlund P: A glycyl radical site in the crystal structure of a class III ribonucleotide reductase. Science 1999;283:1499-1504.
[PubMed]
34.
Logan DT, Mulliez E, Larsson KM, Bodevin S, Atta M, Garnaud PE, Sjoberg BM, Fontecave M: A metal-binding site in the catalytic subunit of anaerobic ribonucleotide reductase. Proc Natl Acad Sci USA 2003;100:3826-3831.
[PubMed]
35.
Martins BM, Blaser M, Feliks M, Ullmann GM, Buckel W, Selmer T: Structural basis for a Kolbe-type decarboxylation catalyzed by a glycyl radical enzyme. J Am Chem Soc 2011;133:14666-14674.
[PubMed]
36.
O'Brien JR, Raynaud C, Croux C, Girbal L, Soucaille P, Lanzilotta WN: Insight into the mechanism of the B12-independent glycerol dehydratase from Clostridium butyricum: preliminary biochemical and structural characterization. Biochemistry 2004;43:4635-4645.
[PubMed]
37.
Ollagnier S, Mulliez E, Schmidt PP, Eliasson R, Gaillard J, Deronzier C, Bergman T, Graslund A, Reichard P, Fontecave M: Activation of the anaerobic ribonucleotide reductase from Escherichia coli. The essential role of the iron-sulfur center for S-adenosylmethionine reduction. J Biol Chem 1997;272:24216-24223.
[PubMed]
38.
Peng Y, Veneziano SE, Gillispie GD, Broderick JB: Pyruvate formate-lyase, evidence for an open conformation favored in the presence of its activating enzyme. J Biol Chem 2010;285:27224-27231.
[PubMed]
39.
Poveda J, Sanchez-Nino MD, Glorieux G, Sanz AB, Egido J, Vanholder R, Ortiz A: p-Cresyl sulphate has pro-inflammatory and cytotoxic actions on human proximal tubular epithelial cells. Nephrol Dial Transplant 2014;29:56-64.
[PubMed]
40.
Sasaki K, Uneyama K, Nagura S: An energetic explanation of the Kolbe electrosynthesis. Electrochim Acta 1966;11:891-894.
41.
Selmer T, Andrei PI: p-Hydroxyphenylacetate decarboxylase from Clostridium difficile. A novel glycyl radical enzyme catalysing the formation of p-cresol. Eur J Biochem 2001;268:1363-1372.
[PubMed]
42.
Selmer T, Pierik AJ, Heider J: New glycyl radical enzymes catalysing key metabolic steps in anaerobic bacteria. Biol Chem 2005;386:981-988.
[PubMed]
43.
Selvaraj B, Pierik AJ, Bill E, Martins BM: 4-Hydroxyphenylacetate decarboxylase activating enzyme catalyses a classical S-adenosylmethionine reductive cleavage reaction. J Biol Inorg Chem 2013;18:633-643.
[PubMed]
44.
Selvaraj B, Pierik AJ, Bill E, Martins BM: The ferredoxin-like domain of the activating enzyme is required for generating a lasting glycyl radical in 4-hydroxyphenylacetate decarboxylase. J Biol Inorg Chem 2014;19:1317-1326.
[PubMed]
45.
Shisler KA, Broderick JB: Glycyl radical activating enzymes: structure, mechanism, and substrate interactions. Arch Biochem Biophys 2014;546:64-71.
[PubMed]
46.
Sofia HJ, Chen G, Hetzler BG, Reyes-Spindola JF, Miller NE: 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 2001;29:1097-1106.
[PubMed]
47.
Stone RW, Machamer HE, Mcaleer WJ, Oakwood TS: Fermentation of tyrosine by marine bacteria. Arch Biochem 1949;21:217-223.
[PubMed]
48.
Uhlin U, Eklund H: Structure of ribonucleotide reductase protein R1. Nature 1994;370:533-539.
[PubMed]
49.
Ullmann RT, Ullmann GM: GMCT: A Monte Carlo simulation package for macromolecular receptors. J Comp Chem 2012;33:887-900.
[PubMed]
50.
Vey JL, Yang J, Li M, Broderick WE, Broderick JB, Drennan CL: Structural basis for glycyl radical formation by pyruvate formate-lyase activating enzyme. Proc Natl Acad Sci USA 2008;105:16137-16141.
[PubMed]
51.
Wong KK, Murray BW, Lewisch SA, Baxter MK, Ridky TW, Ulissidemario L, Kozarich JW: Molecular properties of pyruvate formate-lyase activating enzyme. Biochemistry 1993;32:14102-14110.
[PubMed]
52.
Yang J, Naik SG, Ortillo DO, Garcia-Serres Li RM, Broderick WE, Huynh HB, Broderick JB: The iron-sulfur cluster of pyruvate formate-lyase activating enzyme in whole cells: cluster interconversion and a valence-localized [4Fe-4S]2+ state. Biochemistry 2009;48:9234-9241.
[PubMed]
53.
Yokoyama MT, Carlson JR: Production of skatole and para-cresol by a rumen Lactobacillus Sp. Appl Environ Microbiol 1981;41:71-76.
[PubMed]
54.
Yu L, Blaser M, Andrei PI, Pierik AJ, Selmer T: 4-Hydroxyphenylacetate decarboxylases: properties of a novel subclass of glycyl radical enzyme systems. Biochemistry 2006;45:9584-9592.
[PubMed]
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