4-Hydroxyphenylacetate decarboxylase activating enzyme catalyses a classical S-adenosylmethionine reductive cleavage reaction

• Published in: Journal of biological inorganic chemistry 2013-08, Vol.18 (6), p.633-643
• Main Authors: Selvaraj, Brinda, Pierik, Antonio J., Bill, Eckhard, Martins, Berta M.
• Format: Article
• Language: English
• Subjects: Biocatalysis; Biochemistry; Biomedical and Life Sciences; Carboxy-Lyases - chemistry; Carboxy-Lyases - isolation & purification; Carboxy-Lyases - metabolism; Clostridium - enzymology; Electron Spin Resonance Spectroscopy; Life Sciences; Microbiology; Original Paper; Oxidation-Reduction; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; S-Adenosylmethionine - chemistry; S-Adenosylmethionine - metabolism; Spectroscopy, Mossbauer
• ISSN: 0949-8257; 1432-1327
• DOI: 10.1007/s00775-013-1008-2

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.