title: 4-Hydroxyphenylacetate decarboxylase activating enzyme catalyses a classical S-adenosylmethionine reductive cleavage reaction.
abstract: 4-Hydroxyphenylacetate decarboxylase (4Hpad) is an Fe/S cluster containing glycyl radical enzyme (GRE), which catalyses the last step of tyrosine fermentation in clostridia, generating the bacteriostatic p-cresol. The respective activating enzyme (4Hpad-AE) displays two cysteine-rich motifs in addition to the classical S-adenosylmethionine (SAM) binding cluster (RS cluster) motif. These additional motifs are also present in other glycyl radical activating enzymes (GR-AE) and it has been postulated that these orthologues may use an alternative SAM homolytic cleavage mechanism, generating a putative 3-amino-3-carboxypropyl radical and 5'-deoxy-5'-(methylthio)adenosine but not a 5'-deoxyadenosyl radical and methionine. 4Hpad-AE produced from a codon-optimized synthetic gene binds a maximum of two [4Fe-4S](2+/+) clusters as revealed by EPR and Mössbauer spectroscopy. The enzyme only catalyses the turnover of SAM under reducing conditions, and the reaction products were identified as 5'-deoxyadenosine (quenched form of 5'-deoxyadenosyl radical) and methionine. We demonstrate that the 5'-deoxyadenosyl radical is the activating agent for 4Hpad through p-cresol formation and correlation between the production of 5'-deoxyadenosine and the generation of glycyl radical in 4Hpad. Therefore, we conclude that 4Hpad-AE catalyses a classical SAM-dependent glycyl radical formation as reported for GR-AE without auxiliary clusters. Our observation casts doubt on the suggestion that GR-AE containing auxiliary clusters catalyse the alternative cleavage reaction detected for glycerol dehydratase activating enzyme.
authors.affiliation: Institut für Biologie, Strukturbiologie/Biochemie-Radikalenzyme, Humboldt-Universität zu Berlin, Unter den Linden 6, 10099 Berlin, Germany.
authors.forename: Brinda
authors.initials: B
authors.lastname: Selvaraj
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Pierik
authors.forename: Eckhard
authors.initials: E
authors.lastname: Bill
authors.forename: Berta M
authors.initials: BM
authors.lastname: Martins
mesh_terms: D055162:Biocatalysis; D002262:Carboxy-Lyases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism*; D015204:Spectroscopy, Mossbauer
chemical_list: D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; C425067:4-hydroxyphenylacetate decarboxylase; D002262:Carboxy-Lyases