The Thermus thermophilus DEAD-box helicase Hera consists of the conserved helicase core, followed by a dimerization domain (DD) and an RNA-binding domain (RBD). The RBD mediates high-affinity binding to an RNA hairpin; the DD mediates formation of a stable dimer. In the dimer, the active sites of the two helicase cores face each other in an ideal configuration to cooperate functionally in RNA unwinding. Here, we dissect the communication between the two RBDs and helicase cores by characterizing dimeric deletion variants with two cores, but two, one, or no RBDs, variants with both RBDs, but two, one, or no functional core, and variants with one core and one RBD, either on the same or opposite protomers. We show that RNA binds to Hera in a two-step mechanism, with an initial interaction between the RBD and a hairpin, followed by the interaction of the core with the flanking single- or double-stranded region. The duplex preferentially interacts with the core on the same protomer in the absence of ATP, but in the presence of ATP, interactions with the other core become possible. Overall, our results point to limited but significant cooperativity between the two protomers in RNA unwinding.

The genome of the α-proteobacterium Pseudaminobacter salicylatoxidans codes for a ferrous iron containing ring-fission dioxygenase which catalyzes the 1,2-cleavage of (substituted) salicylate(s), gentisate (2,5-dihydroxybenzoate), and 1-hydroxy-2-naphthoate. Sequence alignments suggested that the "salicylate 1,2-dioxygenase" (SDO) from this strain is homologous to gentisate 1,2-dioxygenases found in bacteria, archaea and fungi. In the present study the catalytic mechanism of the SDO and gentisate 1,2-dioxygenases in general was analyzed based on sequence alignments, mutational and previously performed crystallographic studies and mechanistic comparisons with "extradiol- dioxygenases" which cleave aromatic nuclei in the 2,3-position. Different highly conserved amino acid residues that were supposed to take part in binding and activation of the organic substrates were modified in the SDO by site-specific mutagenesis and the enzyme variants subsequently analyzed for the conversion of salicylate, gentisate and 1-hydroxy-2-naphthoate. The analysis of enzyme variants which carried exchanges in the positions Arg83, Trp104, Gly106, Gln108, Arg127, His162 and Asp174 demonstrated that Arg83 and Arg127 were indispensable for enzymatic activity. In contrast, residual activities were found for variants carrying mutations in the residues Trp104, Gly106, Gln108, His162, and Asp174 and some of these mutants still could oxidize gentisate, but lost the ability to convert salicylate. The results were used to suggest a general reaction mechanism for gentisate-1,2-dioxygenases and to assign to certain amino acid residues in the active site specific functions in the cleavage of (substituted) salicylate(s).

Quercetinases are metal-dependent dioxygenases of the cupin superfamily. While fungal quercetinases are copper proteins, recombinant Streptomyces quercetinase (QueD) was previously described to be capable of incorporating Ni2+ and some other divalent metal ions. This raises the questions of which factors determine metal selection, and which metal ion is physiologically relevant.

The salicylate 1,2‐dioxygenase ( SDO ) from the bacterium P seudaminobacter salicylatoxidans BN 12 is a versatile gentisate 1,2‐dioxygenase ( GDO ) that converts both gentisate (2,5‐dihydroxybenzoate) and various monohydroxylated substrates. Several variants of this enzyme were rationally designed based on the previously determined enzyme structure and sequence differences between the SDO and the ‘conventional’ GDO from Corynebacterium glutamicum . This was undertaken in order to define the structural elements that give the SDO its unique ability to dioxygenolytically cleave (substituted) salicylates. SDO variants M 103 L , G 106 A , G 111 A , R 113 G , S 147 R and F 159 Y were constructed and it was found that G 106 A oxidized only gentisate; 1‐hydroxy‐2‐naphthoate and salicylate were not converted. This indicated that this enzyme variant behaves like previously known ‘conventional’ GDO s. Crystals of the G 106 A SDO variant and its complexes with salicylate and gentisate were obtained under anaerobic conditions, and the structures were solved and analyzed. The amino acid residue Gly106 is located inside the SDO active site cavity but does not directly interact with the substrates. Crystal structures of G 106 A SDO complexes with gentisate and salicylate showed a different binding mode for salicylate when compared with the wild‐type enzyme. Thus, salicylate coordinated in the G 106 A variant with the catalytically active Fe(II) ion in an unusual and unproductive manner because of the inability of salicylate to displace a hydrogen bond that was formed between T rp104 and A sp174 in the G 106 A variant. It is proposed that this type of unproductive substrate binding might generally limit the substrate spectrum of ‘conventional’ GDO s. Database Structural data are available in the Protein Data Bank databases under the accession numbers 3NST , 3NWA , 3NVC

DEAD box helicases catalyze the ATP-dependent destabilization of RNA duplexes. Whereas duplex separation is mediated by the helicase core shared by all members of the family, flanking domains often contribute to binding of the RNA substrate. The Thermus thermophilus DEAD-box helicase Hera (for "heat-resistant RNA-binding ATPase") contains a C-terminal RNA-binding domain (RBD). We have analyzed RNA binding to the Hera RBD by a combination of mutational analyses, nuclear magnetic resonance and X-ray crystallography, and identify residues on helix α1 and the C-terminus as the main determinants for high-affinity RNA binding. A crystal structure of the RBD in complex with a single-stranded RNA resolves the RNA-protein interactions in the RBD core region around helix α1. Differences in RNA binding to the Hera RBD and to the structurally similar RBD of the Bacillus subtilis DEAD box helicase YxiN illustrate the versatility of RNA recognition motifs as RNA-binding platforms. Comparison of chemical shift perturbation patterns elicited by different RNAs, and the effect of sequence changes in the RNA on binding and unwinding show that the RBD binds a single-stranded RNA region at the core and simultaneously contacts double-stranded RNA through its C-terminal tail. The helicase core then unwinds an adjacent RNA duplex. Overall, the mode of RNA binding by Hera is consistent with a possible function as a general RNA chaperone.

Key amino acid residues of the salicylate 1,2-dioxygenase (SDO), an iron (II) class III ring cleaving dioxygenase from Pseudaminobacter salicylatoxidans BN12, were selected, based on amino acid sequence alignments and structural analysis of the enzyme, and modified by site-directed mutagenesis to obtain variant forms with altered catalytic properties. SDO shares with 1-hydroxy-2-naphthoate dioxygenase (1H2NDO) its unique ability to oxidatively cleave monohydroxylated aromatic compounds. Nevertheless SDO is more versatile with respect to 1H2NDO and other known gentisate dioxygenases (GDOs) because it cleaves not only gentisate and 1-hydroxy-2-naphthoate (1H2NC) but also salicylate and substituted salicylates. Several enzyme variants of SDO were rationally designed to simulate 1H2NDO. The basic kinetic parameters for the SDO mutants L38Q, M46V, A85H and W104Y were determined. The enzyme variants L38Q, M46V, A85H demonstrated higher catalytic efficiencies toward 1-hydroxy-2-naphthoate (1H2NC) compared to gentisate. Remarkably, the enzyme variant A85H effectively cleaved 1H2NC but did not oxidize gentisate at all. The W104Y SDO mutant exhibited reduced reaction rates for all substrates tested. The crystal structures of the A85H and W104Y variants were solved and analyzed. The substitution of Ala85 with a histidine residue caused significant changes in the orientation of the loop containing this residue which is involved in the active site closing upon substrate binding. In SDO A85H this specific loop shifts away from the active site and thus opens the cavity favoring the binding of bulkier substrates. Since this loop also interacts with the N-terminal residues of the vicinal subunit, the structure and packing of the holoenzyme might be also affected.

RNA helicases unwind their RNA substrates in an ATP-dependent reaction, and are central to all cellular processes involving RNA. They have important roles in viral life cycles, where RNA helicases are either virus-encoded or recruited from the host. Vertebrate RNA helicases sense viral infections, and trigger the innate antiviral immune response. RNA helicases have been implicated in protozoic, bacterial and fungal infections. They are also linked to neurological disorders, cancer, and aging processes. Genome-wide studies continue to identify helicase genes that change their expression patterns after infection or disease outbreak, but the mechanism of RNA helicase action has been defined for only a few diseases. RNA helicases are prognostic and diagnostic markers and suitable drug targets, predominantly for antiviral and anti-cancer therapies. This review summarizes the current knowledge on RNA helicases in infection and disease, and their growing potential as drug targets.

The crystallographic structures of the adducts of salicylate 1,2-dioxygenase (SDO) with substrates salicylate, gentisate and 1-hydroxy-2-naphthoate, obtained under anaerobic conditions, have been solved and analyzed.This ring fission dioxygenase from the naphthalenesulfonate-degrading bacterium Pseudaminobacter salicylatoxidans BN12, is a homo-tetrameric class III ring-cleaving dioxygenase containing a catalytic Fe(II) ion coordinated by three histidine residues. SDO is markedly different from the known gentisate 1,2-dioxygenases or 1-hydroxy-2-naphthoate dioxygenases, belonging to the same class, because of its unique ability to oxidatively cleave salicylate, gentisate and 1-hydroxy-2-naphthoate.The crystal structures of the anaerobic complexes of the SDO reveal the mode of binding of the substrates into the active site and unveil the residues which are important for the correct positioning of the substrate molecules. Upon binding of the substrates the active site of SDO undergoes a series of conformational changes: in particular Arg127, His162, and Arg83 move to make hydrogen bond interactions with the carboxyl group of the substrate molecules. Unpredicted concerted displacements upon substrate binding are observed for the loops composed of residues 40-43, 75-85, and 192-198 where several aminoacidic residues, such as Leu42, Arg79, Arg83, and Asp194, contribute to the closing of the active site together with the amino-terminal tail (residues 2-15).Differences in substrate specificity are controlled by several residues located in the upper part of the substrate binding cavity like Met46, Ala85, Trp104, and Phe189, although we cannot exclude that the kinetic differences observed could also be generated by concerted conformational changes resulting from amino-acid mutations far from the active site. (C) 2011 Elsevier Inc. All rights reserved.