The structure of Rph, an exoribonuclease from Bacillus anthracis, at 1.7 angstrom resolution

Maturation of tRNA precursors into functional tRNA molecules requires trimming of the primary transcript at both the 5' and 3' ends. Cleavage of nucleotides from the 3' stem of tRNA precursors, releasing nucleotide diphosphates, is accomplished in Bacillus by a phosphate-dependent exoribonuclease, Rph. The crystal structure of this enzyme from B. anthracis has been solved by molecular replacement to a resolution of 1.7 angstrom and refined to an R factor of 19.3%. There is one molecule in the asymmetric unit; the crystal packing reveals the assembly of the protein into a hexamer arranged as a trimer of dimers. The structure shows two sulfate ions bound in the active-site pocket, probably mimicking the phosphate substrate and the phosphate of the 3'-terminal nucleotide of the tRNA precursor. Three other bound sulfate ions point to likely RNA-binding sites

Structure of the Branched Chain Amino Acid and GTP Sensing Global Regulator, CodY, from Bacillus subtilis

CodY is a branched-chain amino acid (BCAA) and GTP sensor, and a global regulator of transcription in low G + C Gram-positive bacteria. It controls the expression of over 100 genes and operons, principally by repressing during growth genes whose products are required for adaptations to nutrient limitation. CodY consists of a GAF domain that binds BCAAs and a winged helix-turn-helix (wHTH) domain that binds to DNA, but the way in which these domains interact and the structural basis of the BCAA-dependence of this interaction are unknown. To gain new insights, we determined the crystal structure of unliganded CodY from Bacillus subtilis revealing a 10-turn alpha-helix linking otherwise discrete GAF and wHTH domains. The structure of CodY in complex with isoleucine revealed a reorganised GAF domain. In both complexes CodY was tetrameric. Size exclusion chromatography with multiangle laser light scattering (SEC-MALLS) experiments showed that CodY is a dimer at concentrations found in bacterial cells. Comparison of structures of dimers of unliganded CodY and CodY-Ile derived from the tetramers showed a splaying of the wHTH domains when Ile was bound; splaying is likely to account for the increased affinity of Ile-bound CodY for DNA. Electrophoretic mobility shift and SEC-MALLS analyses of CodY binding to 19-36 base-pair operator fragment are consistent with isoleucine-dependent binding of two CodY dimers per duplex. The implications of these observations for effector control of CodY activity are discussed

Kinetic and structural analysis of redox-reversible artificial imine reductases

Three artificial imine reductases, constructed via supramolecular anchoring utilising Fe III-azotochelin, a natural siderophore, to bind an iridium-containing catalyst to periplasmic siderophore-binding protein (PBP) scaffolds, have previously been synthesised and subjected to catalytic testing. Despite exhibiting high homology and possessing conserved siderophore anchor coordinating residues, the three artificial metalloenzymes (ArMs) displayed significant variability in turnover frequencies (TOFs). To further understand the catalytic properties of these ArMs, their kinetic behaviour was evaluated with respect to the reduction of three cyclic imines: dihydroisoquinoline, harmaline, and papaverine. Kinetic analyses revealed that all examined ArMs adhere to Michaelis-Menten kinetics, with the most pronounced saturation profile observed for the substrate harmaline. Additionally, molecular docking studies suggested varied hydrogen-bonding interactions between substrates and residues within the artificial binding pocket. Pi-stacking and pi-cation interactions were identified for harmaline and papaverine, corroborating the higher affinity of these substrates for the ArMs in comparison to dihydroisoquinoline. Furthermore, it was demonstrated that multiple cavities are capable of accommodating substrates in close proximity to the catalytic centre, thereby rationalising the moderate enantioselectivity conferred by the unmodified scaffolds

Redox-switchable siderophore anchor enables reversible artificial metalloenzyme assembly

Artificial metalloenzymes that contain protein-anchored synthetic catalysts are attracting increasing interest. An exciting, but still unrealized advantage of non-covalent anchoring is its potential for reversibility and thus component recycling. Here we present a siderophore–protein combination that enables strong but redox-reversible catalyst anchoring, as exemplified by an artificial transfer hydrogenase (ATHase). By linking the iron(iii)-binding siderophore azotochelin to an iridium-containing imine-reduction catalyst that produces racemic product in the absence of the protein CeuE, but a reproducible enantiomeric excess if protein bound, the assembly and reductively triggered disassembly of the ATHase was achieved. The crystal structure of the ATHase identified the residues involved in high-affinity binding and enantioselectivity. While in the presence of iron(iii), the azotochelin-based anchor binds CeuE with high affinity, and the reduction of the coordinated iron(iii) to iron(ii) triggers its dissociation from the protein. Thus, the assembly of the artificial enzyme can be controlled via the iron oxidation state

Viral genome packaging terminase cleaves DNA using the canonical RuvC-like two-metal catalysis mechanism

Bacteriophages and large dsDNA viruses encode sophisticated machinery to translocate their DNA into a preformed empty capsid. An essential part of this machine, the large terminase protein, processes viral DNA into constituent units utilizing its nuclease activity. Crystal structures of the large terminase nuclease from the thermophilic bacteriophage G20c show that it is most similar to the RuvC family of the RNase H-like endonucleases. Like RuvC proteins, the nuclease requires either Mn2+, Mg2+ or Co2+ ions for activity, but is inactive with Zn2+ and Ca2+. High resolution crystal structures of complexes with different metals reveal that in the absence of DNA, only one catalytic metal ion is accommodated in the active site. Binding of the second metal ion may be facilitated by conformational variability, which enables the two catalytic aspartic acids to be brought closer to each other. Structural comparison indicates that in common with the RuvC family, the location of the two catalytic metals differs from other members of the RNase H family. In contrast to a recently proposed mechanism, the available data do not support binding of the two metals at an ultra-short interatomic distance. Thus we postulate that viral terminases cleave DNA by the canonical RuvC-like mechanism

Mimicking salmochelin S1 and the interactions of its Fe(III) complex with periplasmic iron siderophore binding proteins CeuE and VctP

A mimic of the tetradentate stealth siderophore salmochelin S1, was synthesised, characterised and shown to form Fe(III) complexes with ligand-to-metal ratios of 1:1 and 3:2. Circular dichroism spectroscopy confirmed that the periplasmic binding proteins CeuE and VctP of Campylobacter jejuni and Vibrio cholerae, respectively, bind the Fe(III) complex of the salmochelin mimic by preferentially selecting Λ-configured Fe(III) complexes. Intrinsic fluorescence quenching studies revealed that VctP binds Fe(III) complexes of the mimic and structurally-related catecholate ligands, such as enterobactin, bis(2, 3-dihydroxybenzoyl-l-serine) and bis(2, 3-dihydroxybenzoyl)-1, 5-pentanediamine with higher affinity than does CeuE. Both CeuE and VctP display a clear preference for the tetradentate bis(catecholates) over the tris(catecholate) siderophore enterobactin. These findings are consistent with reports that V. cholerae and C. jejuni utilise the enterobactin hydrolysis product bis(2, 3-dihydroxybenzoyl)-O-seryl serine for the acquisition of Fe(III) and suggest that the role of salmochelin S1 in the iron uptake of enteric pathogens merits further investigation

Novel Inhibitory Function of the Rhizomucor miehei Lipase Propeptide and Three-Dimensional Structures of Its Complexes with the Enzyme

Many proteins are synthesized as precursors, with propeptides playing a variety of roles such as assisting in folding or preventing them from being active within the cell. While the precise role of the propeptide in fungal lipases is not completely understood, it was previously reported that mutations in the propeptide region of the Rhizomucor miehei lipase have an influence on the activity of the mature enzyme, stressing the importance of the amino acid composition of this region. We here report two structures of this enzyme in complex with its propeptide, which suggests that the latter plays a role in the correct maturation of the enzyme. Most importantly, we demonstrate that the propeptide shows inhibition of lipase activity in standard lipase assays and propose that an important role of the propeptide is to ensure that the enzyme is not active during its expression pathway in the original host

Structural and functional characterisation of three novel fungal amylases with enhanced stability and pH tolerance

Amylases are probably the best studied glycoside hydrolases and have a huge biotechnological value for industrial processes on starch. Multiple amylases from fungi and microbes are currently in use. Whereas bacterial amylases are well suited for many industrial processes due to their high stability, fungal amylases are recognized as safe and are preferred in the food industry, although they lack the pH tolerance and stability of their bacterial counterparts. Here, we describe three amylases, two of which have a broad pH spectrum extending to pH 8 and higher stability well suited for a broad set of industrial applications. These enzymes have the characteristic GH13 α-amylase fold with a central (β/α)8-domain, an insertion domain with the canonical calcium binding site and a C-terminal β-sandwich domain. The active site was identified based on the binding of the inhibitor acarbose in form of a transglycosylation product, in the amylases from Thamnidium elegans and Cordyceps farinosa. The three amylases have shortened loops flanking the nonreducing end of the substrate binding cleft, creating a more open crevice. Moreover, a potential novel binding site in the C-terminal domain of the Cordyceps enzyme was identified, which might be part of a starch interaction site. In addition, Cordyceps farinosa amylase presented a successful example of using the microseed matrix screening technique to significantly speed-up crystallization