Mark S. Dunstan

The structure and catalytic mechanism of a poly(ADP-ribose) glycohydrolase

Post-translational modification of proteins by poly(ADP-ribosyl)ation regulates many cellular pathways that are critical for genome stability, including DNA repair, chromatin structure, mitosis and apoptosis. Poly(ADP-ribose) (PAR) is composed of repeating ADP-ribose units linked via a unique glycosidic ribose–ribose bond, and is synthesized from NAD by PAR polymerases. PAR glycohydrolase (PARG) is the only protein capable of specific hydrolysis of the ribose–ribose bonds present in PAR chains; its deficiency leads to cell death. Here we show that filamentous fungi and a number of bacteria possess a divergent form of PARG that has all the main characteristics of the human PARG enzyme. We present the first PARG crystal structure (derived from the bacterium Thermomonospora curvata), which reveals that the PARG catalytic domain is a distant member of the ubiquitous ADP-ribose-binding macrodomain family. High-resolution structures of T. curvata PARG in complexes with ADP-ribose and the PARG inhibitor ADP-HPD, complemented by biochemical studies, allow us to propose a model for PAR binding and catalysis by PARG. The insights into the PARG structure and catalytic mechanism should greatly improve our understanding of how PARG activity controls reversible protein poly(ADP-ribosyl)ation and potentially of how the defects in this regulation are linked to human disease.

Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation.

Organohalide chemistry underpins many industrial and agricultural processes, and a large proportion of environmental pollutants are organohalides. Nevertheless, organohalide chemistry is not exclusively of anthropogenic origin, with natural abiotic and biological processes contributing to the global halide cycle. Reductive dehalogenases are responsible for biological dehalogenation in organohalide respiring bacteria, with substrates including polychlorinated biphenyls or dioxins. Reductive dehalogenases form a distinct subfamily of cobalamin (B12)-dependent enzymes that are usually membrane associated and oxygen sensitive, hindering detailed studies. Here we report the characterization of a soluble, oxygen-tolerant reductive dehalogenase and, by combining structure determination with EPR (electron paramagnetic resonance) spectroscopy and simulation, show that a direct interaction between the cobalamin cobalt and the substrate halogen underpins catalysis. In contrast to the carbon–cobalt bond chemistry catalysed by the other cobalamin-dependent subfamilies, we propose that reductive dehalogenases achieve reduction of the organohalide substrate via halogen–cobalt bond formation. This presents a new model in both organohalide and cobalamin (bio)chemistry that will guide future exploitation of these enzymes in bioremediation or biocatalysis.

Reengineering orthogonally selective riboswitches

The ability to independently control the expression of multiple genes by addition of distinct small-molecule modulators has many applications from synthetic biology, functional genomics, pharmaceutical target validation, through to gene therapy. Riboswitches are relatively simple, small-molecule–dependent, protein-free, mRNA genetic switches that are attractive targets for reengineering in this context. Using a combination of chemical genetics and genetic selection, we have developed riboswitches that are selective for synthetic “nonnatural” small molecules and no longer respond to the natural intracellular ligands. The orthogonal selectivity of the riboswitches is also demonstrated in vitro using isothermal titration calorimetry and x-ray crystallography. The riboswitches allow highly responsive, dose-dependent, orthogonally selective, and dynamic control of gene expression in vivo. It is possible that this approach may be further developed to reengineer other natural riboswitches for application as small-molecule responsive genetic switches in both prokaryotes and eukaryotes.

Synthesis and Biological Evaluation of Coumarin-Based Inhibitors of NAD(P)H: Quinone Oxidoreductase-1 (NQO1)†

The synthesis is reported here of two novel series of inhibitors of human NAD(P)H quinone oxidoreductase-1 (NQO1), an enzyme overexpressed in several types of tumor cell. The first series comprises substituted symmetric dicoumarol analogues; the second series contains hybrid compounds where one 4-hydroxycoumarin system is replaced by a different aromatic moiety. Several compounds show equivalent or improved NQO1 inhibition over dicoumarol, both in the presence and in the absence of added protein. Further, correlation is demonstrated between the ability of these agents to inhibit NQO1 and computed binding affinity. We have solved the crystal structure of NQO1 complexed to a hybrid compound and find good agreement with the in silico model. For both MIA PaCa-2 pancreatic tumor cells and HCT116 colon cancer cells, dicoumarol shows the greatest toxicity of all compounds. Thus, we provide a computational, synthetic, and biological platform to generate competitive NQO1 inhibitors with superior pharmacological properties to dicoumarol. This will allow a more definitive study of NQO1 activity in cells, in particular, its drug activating/detoxifying properties and ability to modulate oncoprotein stability.

Visualization of poly(ADP-ribose) bound to PARG reveals inherent balance between exo- and endo-glycohydrolase activities

Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo-glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo-mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo-glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo-mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios.

Modular riboswitch toolsets for synthetic genetic control in diverse bacterial species.

Ligand-dependent control of gene expression is essential for gene functional analysis, target validation, protein production, and metabolic engineering. However, the expression tools currently available are difficult to transfer between species and exhibit limited mechanistic diversity. Here we demonstrate how the modular architecture of purine riboswitches can be exploited to develop orthogonal and chimeric switches that are transferable across diverse bacterial species, modulating either transcription or translation, to provide tunable activation or repression of target gene expression, in response to synthetic non-natural effector molecules. Our novel riboswitch-ligand pairings are shown to regulate physiologically important genes required for bacterial motility in Escherichia coli and cell morphology in Bacillus subtilis. These findings are relevant for future gene function studies and antimicrobial target validation, while providing new modular and orthogonal regulatory components for deployment in synthetic biology regimes.

Heterologous expression, purification and cofactor reconstitution of the reductive dehalogenase PceA from Dehalobacter restrictus.

Organohalide respiration is used by a limited set of anaerobic bacteria to derive energy from the reduction of halogenated organic compounds. The enzymes that catalyze the reductive dehalogenation reaction, the reductive dehalogenases, represent a novel and distinct class of cobalamin and Fe-S cluster dependent enzymes. Until now, biochemical studies on reductive dehalogenases have been hampered by the lack of a reliable protein source. Here we present an efficient and robust heterologous production system for the reductive dehalogenase PceA from Dehalobacter restrictus. Large quantities of Strep-tagged PceA fused to a cold-shock induced trigger factor could be obtained from Escherichia coli. The recombinant enzyme was conveniently purified in milligram quantities under anaerobic conditions by StrepTactin affinity chromatography, and the trigger factor could be removed through limited proteolysis. Characterization of the purified PceA by UV-Vis and electron paramagnetic resonance (EPR) spectroscopy reveal that the recombinant protein binds methylcobalamin in the base-on form after proteolytic cleavage of the trigger factor, and that 4Fe-4S clusters can be chemically reconstituted under anoxic conditions. This study demonstrates a novel PceA production platform that allows further study of this new enzyme class.

Structure of the Thiostrepton Resistance Methyltransferase·S-Adenosyl-l-methionine Complex and Its Interaction with Ribosomal RNA

The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr)·S-adenosyl-l-methionine (AdoMet) complex was determined at 2.45-Å resolution. Tsr is definitively confirmed as a Class IV methyltransferase of the SpoU family with an N-terminal “L30-like” putative target recognition domain. The structure and our in vitro analysis of the interaction of Tsr with its target domain from 23 S ribosomal RNA (rRNA) demonstrate that the active biological unit is a Tsr homodimer. In vitro methylation assays show that Tsr activity is optimal against a 29-nucleotide hairpin rRNA though the full 58-nucleotide L11-binding domain and intact 23 S rRNA are also effective substrates. Molecular docking experiments predict that Tsr·rRNA binding is dictated entirely by the sequence and structure of the rRNA hairpin containing the A1067 target nucleotide and is most likely driven primarily by large complementary electrostatic surfaces. One L30-like domain is predicted to bind the target loop and the other is near an internal loop more distant from the target site where a nucleotide change (U1061 to A) also decreases methylation by Tsr. Furthermore, a predicted interaction with this internal loop by Tsr amino acid Phe-88 was confirmed by mutagenesis and RNA binding experiments. We therefore propose that Tsr achieves its absolute target specificity using the N-terminal domains of each monomer in combination to recognize the two distinct structural elements of the target rRNA hairpin such that both Tsr subunits contribute directly to the positioning of the target nucleotide on the enzyme.

Epoxyqueuosine Reductase Structure Suggests a Mechanism for Cobalamin-dependent tRNA Modification

Background: Little is known about epoxyqueuosine reductase (QueG), which catalyzes the final step in the biosynthesis of queuosine. Results: We report solution and structural characterization of Streptococcus thermophilus QueG. Conclusion: The QueG similarity to reductive dehalogenases is largely limited to cofactor binding. Significance: Our study establishes the link between cobalamin-metabolism and tRNA modification and suggests a mechanism for cobalamin-dependent epoxide reduction. Queuosine (Q) is a hypermodified RNA base that replaces guanine in the wobble positions of 5′-GUN-3′ tRNA molecules. Q is exclusively made by bacteria, and the corresponding queuine base is a micronutrient salvaged by eukaryotic species. The final step in Q biosynthesis is the reduction of the epoxide precursor, epoxyqueuosine, to yield the Q cyclopentene ring. The epoxyqueuosine reductase responsible, QueG, shares distant homology with the cobalamin-dependent reductive dehalogenase (RdhA), however the role played by cobalamin in QueG catalysis has remained elusive. We report the solution and structural characterization of Streptococcus thermophilus QueG, revealing the enzyme harbors a redox chain consisting of two [4Fe-4S] clusters and a cob(II)alamin in the base-off form, similar to RdhAs. In contrast to the shared redox chain architecture, the QueG active site shares little homology with RdhA, with the notable exception of a conserved Tyr that is proposed to function as a proton donor during reductive dehalogenation. Docking of an epoxyqueuosine substrate suggests the QueG active site places the substrate cyclopentane moiety in close proximity of the cobalt. Both the Tyr and a conserved Asp are implicated as proton donors to the epoxide leaving group. This suggests that, in contrast to the unusual carbon-halogen bond chemistry catalyzed by RdhAs, QueG acts via Co-C bond formation. Our study establishes the common features of Class III cobalamin-dependent enzymes, and reveals an unexpected diversity in the reductive chemistry catalyzed by these enzymes.

Structures of carboxylic acid reductase reveal domain dynamics underlying catalysis

Carboxylic acid reductase (CAR) catalyzes the ATP- and NADPH-dependent reduction of carboxylic acids to the corresponding aldehydes. The enzyme is related to the non-ribosomal peptide synthetases, consisting of an adenylation domain fused via a peptidyl carrier protein (PCP) to a reductase termination domain. Crystal structures of the CAR adenylation–PCP didomain demonstrate that large-scale domain motions occur between the adenylation and thiolation states. Crystal structures of the PCP–reductase didomain reveal that phosphopantetheine binding alters the orientation of a key Asp, resulting in a productive orientation of the bound nicotinamide. This ensures that reduction of the aldehyde product does not occur. Combining crystallography with small-angle x-ray scattering (SAXS), we propose that molecular interactions between initiation and termination domains are limited to competing PCP docking sites. This is supported by the fact that (R)-pantetheine can support CAR activity for mixtures of the isolated domains. Our model suggests directions for further development of CAR as a biocatalyst.