SummaryPhotoreceptor proteins enable organisms to sense and respond to light. The newly discovered CarH-type photoreceptors use a vitamin B12 derivative, adenosylcobalamin, as the light-sensing chromophore to mediate light-dependent gene regulation. Here, we present crystal structures of Thermus thermophilus CarH in all three relevant states: in the dark, both free and bound to operator DNA, and after light exposure. These structures provide a visualization of how adenosylcobalamin mediates CarH tetramer formation in the dark, how this tetramer binds to the promoter −35 element to repress transcription, and how light exposure leads to a large-scale conformational change that activates transcription. In addition to the remarkable functional repurposing of adenosylcobalamin from an enzyme cofactor to a light sensor, we find that nature also repurposed two independent protein modules in assembling CarH. These results expand the biological role of vitamin B12 and provide fundamental insight into a new mode of light-dependent gene regulation.

Ribonucleotide reductase (RNR) converts ribonucleotides to deoxyribonucleotides, a reaction that is essential for DNA biosynthesis and repair. This enzyme is responsible for reducing all four ribonucleotide substrates, with specificity regulated by the binding of an effector to a distal allosteric site. In all characterized RNRs, the binding of effector dATP alters the active site to select for pyrimidines over purines, whereas effectors dGTP and TTP select for substrates ADP and GDP, respectively. Here, we have determined structures of Escherichia coli class Ia RNR with all four substrate/specificity effector-pairs bound (CDP/dATP, UDP/dATP, ADP/dGTP, GDP/TTP) that reveal the conformational rearrangements responsible for this remarkable allostery. These structures delineate how RNR ‘reads’ the base of each effector and communicates substrate preference to the active site by forming differential hydrogen bonds, thereby maintaining the proper balance of deoxynucleotides in the cell.DOI: http://dx.doi.org/10.7554/eLife.07141.001

TsrM methylates C2 of the indole ring of L-tryptophan (Trp) during the biosynthesis of the quinaldic acid moiety of thiostrepton. It is annotated as a cobalamin-dependent radical S -adenosylmethionine (SAM) methylase; however, TsrM does not reductively cleave SAM to the universal 5ʹ-deoxyadenosyl 5ʹ-radical intermediate, a hallmark of radical-SAM (RS) enzymes. Herein, we report structures of TsrM from Kitasatospora setae , the first of a cobalamin-dependent radical SAM methylase. Unexpectedly, the structures show an essential arginine residue that resides in the proximal coordination sphere of the cobalamin cofactor and a [4Fe–4S] cluster that is ligated by a glutamyl residue and three cysteines in a canonical CxxxCxxC RS motif. Structures in the presence of substrates suggest a substrate-assisted mechanism of catalysis, wherein the carboxylate group of SAM serves as a general base to deprotonate N1 of the tryptophan substrate, facilitating formation of a C2 carbanion.

Edited by Joseph M. Jez This work was supported by National Institutes of Health Grants R35 GM126982 (to C. L. D.) and R01 GM084998 (C. L. F. M.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Figs. S1-S13 and Tables S1-S4. The atomic coordinates and structure factors (codes 6OUV and 6OUW) have been deposited in the Protein Data Bank (http://wwpdb.org/).

No abstract

Octocorals are major contributors of terpenoid chemical diversity in the ocean. Natural products from other sessile marine animals are primarily biosynthesized by symbiotic microbes rather than by the host. Here we challenge this long-standing paradigm by describing a monophyletic lineage of animal-encoded terpene cyclases (TCs), ubiquitous in octocorals. We characterized 15 TC enzymes from nine genera, several of which produce precursors of iconic, coral-specific terpenoids such as pseudopterosin, lophotoxin and eleutherobin. X-ray crystallography reveals that coral TCs share conserved active site residues and structural features with bacterial TCs. The identification of coral TCs enabled the targeted identification of the enzyme that constructs the coral-exclusive capnellane scaffold. Several TC genes are co-localized with genes that encode enzymes known to modify terpenes. This work presents the first example of biosynthetic capacity in the kingdom Animalia that rivals the chemical complexity generated by plants, unlocking the biotechnological potential of octocorals for biomedical applications.

2-Oxoacid:ferredoxin oxidoreductases (OFORs) are essential enzymes in microbial one-carbon metabolism. They use thiamine pyrophosphate to reversibly cleave carbon-carbon bonds, generating low potential (~ −500 mV) electrons. Crystallographic analysis of a recently discovered OFOR, an oxalate oxidoreductase (OOR), has provided a second view of OFOR architecture and active site composition. Using these recent structural data along with the previously determined structures of pyruvate:ferredoxin oxidoreductase, structure-function relationships in this superfamily have been expanded and re-evaluated. Additionally, structural motifs have been defined that better serve to distinguish one OFOR subfamily from another and potentially uncover novel OFORs.

Ribonucleotide reductases (RNRs) convert ribonucleotides to deoxynucleotides, a process essential for DNA biosynthesis and repair. Class Ia RNRs require two dimeric subunits for activity: an α subunit that houses the active site and allosteric regulatory sites and a β subunit that houses the diferric tyrosyl radical cofactor. Ribonucleotide reduction requires that both subunits form a compact αβ state allowing for radical transfer from β to α RNR activity is regulated allosterically by dATP, which inhibits RNR, and by ATP, which restores activity. For the well-studied class Ia RNR, dATP binding to an allosteric site on α promotes formation of an αβ ring-like state. Here, we investigate whether the αβ formation causes or results from RNR inhibition. We demonstrate that substitutions at the α-β interface (S37D/S39A-α, S39R-α, S39F-α, E42K-α, or L43Q-α) that disrupt the αβ oligomer abrogate dATP-mediated inhibition, consistent with the idea that αβ formation is required for dATP's allosteric inhibition of RNR. Our results further reveal that the α-β interface in the inhibited state is highly sensitive to manipulation, with a single substitution interfering with complex formation. We also discover that residues at the α-β interface whose substitution has previously been shown to cause a mutator phenotype in ( S39F-α or E42K-α) are impaired only in their activity regulation, thus linking this phenotype with the inability to allosterically down-regulate RNR. Whereas the cytotoxicity of RNR inhibition is well-established, these data emphasize the importance of down-regulation of RNR activity.