In a conventional class I ribonucleotide reductase (RNR), a diiron(II/II) cofactor in the R2 subunit reacts with oxygen to produce a diiron(III/IV) intermediate, which generates a stable tyrosyl radical (Y*). The Y* reversibly oxidizes a cysteine residue in the R1 subunit to a cysteinyl radical (C*), which abstracts the 3'-hydrogen of the substrate to initiate its reduction. The RNR from Chlamydia trachomatis lacks the Y*, and it had been proposed that the diiron(III/IV) complex in R2 directly generates the C* in R1. By enzyme activity measurements and spectroscopic methods, we show that this RNR actually uses a previously unknown stable manganese(IV)/iron(III) cofactor for radical initiation.

Tyrosine hydroxylase (TyrH 1), the key enzyme in the biosynthesis of catecholamine neurotransmitters, is one of three members of the aromatic amino acid hydroxylase enzyme family. 2,3 The enzyme is found in the brain and adrenal gland where it catalyses the conversion of L-tyrosine to L-DOPA. The other members of the family are phenylalanine hydroxylase, which catabolizes excess phenylalanine to tyrosine, and tryptophan hydroxylase, which catalyzes the rate limiting step in the biosynthesis of the neurotransmitter serotonin. All three enzymes have a mononuclear non-heme iron, coordinated by the common His 2-Glu facial triad motif, 4,5 and use a tetrahydropterin to activate dioxygen for hydroxylation of the aromatic side chains of their corresponding amino acid substrates. 2,3 In the proposed mechanism 6-8 (Scheme 1), oxygen reacts with ferrous iron and tetrahydropterin to produce a Fe(IV)O (ferryl) hydroxylating intermediate and 4a-hydroxypterin (4a-HOPH 3). Then, through an electrophilic aromatic substitution, the ferryl species reacts with the aromatic side chain of the tyrosine substrate (Tyr) to form the product dihydroxyphenylalanine (DOPA). To date there has been no direct evidence for this ferryl species. Here, we report the detection of an Fe(IV) intermediate, which is likely to be the proposed ferryl species, in the TyrH reaction by the use of rapid reaction methods. The anaerobic TyrH•Fe(II) •6-MePH 4 •Tyr complex 9 was reacted with oxygen and quenched by rapid-freeze at time points from 20 ms to 390 ms. 10 Figure 1 (left panel) shows representative Mössbauer spectra of the samples from such a time course. The spectrum of the reactant complex reveals the presence of two broad lines with parameters typical of high-spin Fe(II). The asymmetry suggests the presence of at least two distinct Fe(II) complexes. A new line at ~0.9 mm/s is observed in the spectra of samples in which the reactant complex was exposed to oxygen for either 20 ms or 100 ms, but it is not detected in the spectrum of a sample reacted for 390 ms. Thus, this peak is associated with a reaction intermediate which exhibits a quadrupole doublet in a weak external magnetic field. The low-energy line of this quadrupole doublet overlaps with the low-energy line of the Fe(II). The features of the intermediate are similar to those observed for Fe(IV) intermediates in other mononuclear non-heme enzymes. 11,12

Cytosine residues in mammalian DNA occur in five forms, cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The ten-eleven translocation (Tet) dioxygenases convert 5mC to 5hmC, 5fC and 5caC in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions1–4. The Tet family of dioxygenases is widely distributed across the tree of life5, including the heterolobosean amoeboflagellate Naegleria gruberi. The genome of Naegleria6 encodes homologs of mammalian DNA methyltransferase and Tet proteins7. Here we study biochemically and structurally one of the Naegleria Tet-like proteins (NgTet1), which shares significant sequence conservation (approximately 14% identity or 39% similarity) with mammalian Tet1. Like mammalian Tet proteins, NgTet1 acts on 5mC and generates 5hmC, 5fC and 5caC. The crystal structure of NgTet1 complexed with DNA containing a 5mCpG site revealed that NgTet1 uses a base-flipping mechanism to access 5mC. The DNA is contacted from the minor groove and bent towards the major groove. The flipped 5mC is positioned in the active site pocket with planar stacking contacts, Watson–Crick polar hydrogen bonds and van der Waals interactions specific for 5mC. The sequence conservation between NgTet1 and mammalian Tet1, including residues involved in structural integrity and functional significance, suggests structural conservation across phyla.

Analysis of the spectroscopic signatures of the R2-W48F/D84E biferric peroxo intermediate identifies a cis mu-1,2 peroxo coordination geometry. DFT geometry optimizations on both R2-W48F/D84E and R2-wild-type peroxo intermediate models including constraints imposed by the protein also identify the cis mu-1,2 peroxo geometry as the most stable peroxo intermediate structure. This study provides significant insight into the electronic structure and reactivity of the R2-W48F/D84E peroxo intermediate, structurally related cis mu-1,2 peroxo model complexes, and other enzymatic biferric peroxo intermediates.

Bisulfite sequencing detects 5mC and 5hmC at single-base resolution. However, bisulfite treatment damages DNA, which results in fragmentation, DNA loss, and biased sequencing data. To overcome these problems, enzymatic methyl-seq (EM-seq) was developed. This method detects 5mC and 5hmC using two sets of enzymatic reactions. In the first reaction, TET2 and T4-BGT convert 5mC and 5hmC into products that cannot be deaminated by APOBEC3A. In the second reaction, APOBEC3A deaminates unmodified cytosines by converting them to uracils. Therefore, these three enzymes enable the identification of 5mC and 5hmC. EM-seq libraries were compared with bisulfite-converted DNA, and each library type was ligated to Illumina adaptors before conversion. Libraries were made using NA12878 genomic DNA, cell-free DNA, and FFPE DNA over a range of DNA inputs. The 5mC and 5hmC detected in EM-seq libraries were similar to those of bisulfite libraries. However, libraries made using EM-seq outperformed bisulfite-converted libraries in all specific measures examined (coverage, duplication, sensitivity, etc.). EM-seq libraries displayed even GC distribution, better correlations across DNA inputs, increased numbers of CpGs within genomic features, and accuracy of cytosine methylation calls. EM-seq was effective using as little as 100 pg of DNA, and these libraries maintained the described advantages over bisulfite sequencing. EMseq library construction, using challenging samples and lower DNA inputs, opens new avenues for research and clinical applications.

Nuclear gamma resonance spectroscopy, also known as Mössbauer spectroscopy, is a technique that probes transitions between the nuclear ground state and a low-lying nuclear excited state. The nucleus most amenable to Mössbauer spectroscopy is 57Fe, and 57Fe Mössbauer spectroscopy provides detailed information about the chemical environment and electronic structure of iron. Iron is by far the most structurally and functionally diverse metal ion in biology, and 57Fe Mössbauer spectroscopy has played an important role in the elucidation of its biochemistry. In this article, we give a brief introduction to the technique and then focus on two recent exciting developments pertaining to the application of 57Fe Mössbauer spectroscopy in biochemistry. The first is the use of the rapid freeze-quench method in conjunction with Mössbauer spectroscopy to monitor changes at the Fe site during a biochemical reaction. This method has allowed for trapping and subsequent detailed spectroscopic characterization of reactive intermediates and thus has provided unique insight into the reaction mechanisms of Fe-containing enzymes. We outline the methodology using two examples: (1) oxygen activation by the non-heme diiron enzymes and (2) oxygen activation by taurine:alpha-ketoglutarate dioxygenase (TauD). The second development concerns the calculation of Mössbauer parameters using density functional theory (DFT) methods. By using the example of TauD, we show that comparison of experimental Mössbauer parameters with those obtained from calculations on model systems can be used to provide insight into the structure of a reaction intermediate.

RimO, encoded by the yliG gene in Escherichia coli, has been recently identified in vivo as the enzyme responsible for the attachment of a methylthio group on the β-carbon of Asp88 of the small ribosomal protein S12 [Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. USA, 105, 1826USA, 105, -1831. To date, it is the only enzyme known to catalyze methylthiolation of a protein substrate; the four other naturally occurring methylthio modifications have been observed on tRNA. All members of the methylthiotransferase (MTTase) family, to which RimO belongs, have been shown to contain the canonical CxxxCxxC motif in their primary structures that is typical of the radical S-adenosylmethionine (SAM) family of proteins. MiaB, the only characterized MTTase, and the enzyme experimentally shown to be responsible for methylthiolation of N 6 -isopentenyladenosine of tRNA in E. coli and Thermotoga maritima, has been demonstrated to harbor two distinct clusters. Herein, we report in vitro biochemical, and spectroscopic characterization of RimO. We show by analytical and spectroscopic methods that RimO, heterologously overproduced in E. coli in the presence of iron-sulfur cluster biosynthesis proteins from Azotobacter vinelandii, contains one [4Fe-4S] 2+ cluster. Reconstitution of this form of RimO (RimO rcn ) with 57 Fe and sodium sulfide results in a protein that contains two [4Fe-4S] 2+ clusters, similar to MiaB. We also show by mass spectrometry that RimO rcn catalyzes the attachment of a methylthio group to a peptide substrate analog that mimics the loop structure bearing aspartyl 88 of the S12 ribosomal protein from E. coli. Kinetic analysis of this reaction shows that the activity of RimO rcn in the presence of the substrate analog does not support a complete turnover. We discuss the possible requirement for an assembled ribosome for fully active RimO in vitro. Our findings are † This work was supported by NIH Grant GM-63847 and NSF Grant MCB-0133826 (S.J.B.), the Dreyfus Foundation (Teacher Scholar Award to C.K.), the Beckman Foundation (Young Investigator Award to C.K.), and New England Biolabs. * To whom correspondence should be addressed. Squire J. Booker, 104 Chemistry Building, The Pennsylvania State University, University Park, PA 16802. Phone: 814-865-8793. Fax: 814-865-2927. Squire@psu.edu. Carsten Krebs, 104 Chemistry Building, The Pennsylvania State University, University Park, PA 16802. Phone: 814-865-6089. Fax: 814-865-2927. ckrebs@psu.edu. Lana Saleh, 240 County Road, Ipswich, MA 01908. Phone: 978-380-7446. Fax: 978-921-1350. Saleh@neb.com. Δ These authors contributed equally to this work 3 The EPR and Mössbauer spectroscopic features of RimO ai are consistent with the presence of a small amount of the SAM-bound form, because the spectral features of the SAM-bound and SAM-free forms overlap heavily. Supporting Information AvailableFigures S1, S2, S3, S4, S5, and S6. This material is available free of charge via the Internet at http...

Ribosomal protein S12 undergoes a unique posttranslational modification, methylthiolation of residue D88, in Escherichia coli and several other bacteria. Using mass spectrometry, we have identified the enzyme responsible for this modification in E. coli, the yliG gene product. This enzyme, which we propose be called RimO, is a radical-S-adenosylmethionine protein that bears strong sequence similarity to MiaB, which methylthiolates tRNA. We show that RimO and MiaB represent two of four subgroups of a larger, ancient family of likely methylthiotransferases, the other two of which are typified by Bacillus subtilis YqeV and Methanococcus jannaschii Mj0867, and we predict that RimO is unique among these subgroups in its modification of protein as opposed to tRNA. Despite this, RimO has not significantly diverged from the other three subgroups at the sequence level even within the C-terminal TRAM domain, which in the methyltransferase RumA is known to bind the RNA substrate and which we presume to be responsible for substrate binding and recognition in all four subgroups of methylthiotransferases. To our knowledge, RimO and MiaB represent the most extreme known case of resemblance between enzymes modifying protein and nucleic acid. The initial results presented here constitute a bioinformatics-driven prediction with preliminary experimental validation that should serve as the starting point for several interesting lines of further inquiry.methylthiotransferase ͉ posttranslational modification ͉ radical-SAM protein T he translational apparatus undergoes numerous posttranscriptional and posttranslational modifications of its RNA and protein components, respectively, and the enzymes responsible for many of these modifications have been identified in recent years (1, 2). One modification for which the responsible enzyme is currently unknown is the methylthiolation of the ␤-carbon of residue D88 of ribosomal protein S12 in Escherichia coli (Fig. 1A) (3). Intriguingly, D88 is strictly conserved in all S12 homologs from bacteria, archaea, and eukaryotes. No spontaneous D88 mutants have been identified, and attempts to alter the residue by site-directed mutagenesis have failed where mutation of surrounding residues succeeded (4), suggesting that this aspartic acid residue serves an essential function. The methylthiol modification has also been observed in S12 from Rhodopseudomonas palustris and Thermus thermophilus (3,5,6), but it appears not to be present in cytoplasmic ribosomes from rat and human cells (7,8). In T. thermophilus, S12 P90R and P90W mutants have been shown to be deficient in the D88 modification, presumably due to steric hindrance of the modifying enzyme by the bulky sidechains (4). Thus, although D88 itself appears to be universal and essential, the methylthiol modification is not.The function of this modification remains unclear, but its location suggests a possible role in decoding or translocation. The solved crystal structure of S12 from T. thermophilus reveals that D88 sits within one of two highly cons...