• 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • br Experimental section br Acknowledgments MIN cells were ki


    Experimental section
    Acknowledgments MIN6 cells were kindly provided by Dr. Junichi Miyazaki, Osaka University. This research was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan (JP16H05099 and JP18H04609 to K.H., and JP16H06574 to T.U.), SENTAN, JST to K.H, and Hirosaki University Institutional Grant to K.Y. K.H. was also supported by a grant JSPS Core-to-Core program, A. Advanced Research Networks.
    Introduction Radical S-adenosylmethionine (SAM) enzymes are a large superfamily of enzymes that utilize radical chemistry to catalyze diverse reactions through a similar mechanism for radical initiation. The radical SAM enzymes utilize a [4Fe–4S] cluster that is coordinated to the enzyme via a conserved cysteine motif, most commonly CX3CX2C, that coordinates three of the four irons of the cluster. The fourth iron of the cluster is then free to bind SAM through its amino and carboxylate moieties (Fig. 1). In the reduced state, the [4Fe–4S]+ cluster transfers an electron to SAM, resulting in homolytic cleavage of SAM to produce methionine and the highly reactive 5′-deoxyadenosyl radical (dAdo·) intermediate. The dAdo· abstracts a hydrogen energy metabolism from substrate to produce 5′-deoxyadenosine (dAdoH) and a substrate radical (Fig. 2, blue arrow) which can be the product of the reaction or can undergo further transformation [1], [2], [3]. In addition to a common mechanism, the radical SAM enzymes exhibit a conserved fold, with the [4Fe–4S] cluster bound within a partial (α/β)6 or full (α/β)8 triosephophate isomerase (TIM) barrel (Fig. 3) [1]. Other variations of the cluster binding motif [4], [5] and enzyme fold [6], [7] have been indentified in radical SAM enzymes or radical SAM-like enzymes. SAM has also been reported to undergo alternative cleavage reactions: in a radical SAM-like enzyme [6], [7] as well as one GRE–AE [8], cleavage of the SC(γ) bond has been reported (Fig. 2, green arrow), while the radical SAM enzyme TsrM cleaves the SC(methyl) bond of SAM but in a non-radical mechanism [9]. This review will focus on the radical SAM enzyme pyruvate formate lyase activating enzyme (PFL-AE) as well as other radical SAM enzymes that utilize SAM to abstract a hydrogen atom from a protein glycine residue, placing them in a group known as glycyl radical enzyme activating enzymes (GRE–AEs).
    The glycyl radical enzymes: substrates for the GRE–AEs The GRE–AEs are a subclass of radical SAM enzymes which, after the production of the dAdo·, abstract a hydrogen atom from the alpha carbon of a highly conserved glycine residue in the enzymes known as glycyl radical enzymes (GREs). The resulting glycyl radical is catalytically essential for the GRE, and during GRE catalysis, it abstracts an H-atom from a conserved cysteine residue to produce a thiyl radical followed by generation of a substrate radical (Fig. 4). The GREs include pyruvate formate lyase (PFL) [10], [11], [12], [13], [14], [15], anaerobic ribonucleotide reductase (aRNR) [16], [17], [18], [19], [20], [21], benzylsuccinate synthase (Bss) [22], [23], [24], [25], [26], B12-independent glycerol dehydratase (Gdh) [27], [28], [29], 4-hydroxyphenylacetate energy metabolism decarboxylase (Hpd) [30], [31], [32], and CutC (more recently named choline trimethylamine-lyase or choline TMA-lyase) [33], [34]. Each of these enzymes have a specific activating enzyme, and current results indicate no cross-reactivity between the GRE–AE and any non-partner GRE. Although the GREs catalyze a diverse set of reactions, they share considerable sequence and structural homology. They are most commonly homodimeric proteins with a subunit size of 80–100kDa, although Bss and Hpd contain additional subunits [25], [35], [36]. The core structure of a GRE monomer consists of a 10-stranded β-barrel surrounded by α-helices (Fig. 5) [10], [11], [20], [28], [30], [37]. The GREs have half-site reactivity where only one monomer is activated by its activating enzyme with the catalytic glycine residing on a finger loop with a highly conserved motif, RVXG[FWY]X6–8[FL]X4QX2[IV]X2R [36] and is in close proximity to the active site cysteine in the crystallized inactive state (Fig. 6). The glycyl radical produced during activation is highly stable under anaerobic conditions, with a half life of more than 24h in the case of PFL [38], [39]. The radical is then transferred to a conserved cysteine, or two cysteines sequentially in the case of PFL [13], [40], whereupon this thiyl radical abstracts an H-atom from substrate to produce a substrate radical. Product is then formed through re-abstraction of an H-atom to reproduce the thiyl radical (Fig. 4). Further details on individual GREs and their cognate activating enzymes are provided in the following sections.