RecO is a recombination mediator protein (RMP) important for homologous recombination, replication repair and DNA annealing in bacteria. In all pathways, the single-stranded (ss) DNA binding protein, SSB, plays an inhibitory role by protecting ssDNA from annealing and recombinase binding. Conversely, SSB may stimulate each reaction through direct interaction with RecO. We present a crystal structure of Escherichia coli RecO bound to the conserved SSB C-terminus (SSB-Ct). SSB-Ct binds the hydrophobic pocket of RecO in a conformation similar to that observed in the ExoI/SSB-Ct complex. Hydrophobic interactions facilitate binding of SSB-Ct to RecO and RecO/RecR complex in both low and moderate ionic strength solutions. In contrast, RecO interaction with DNA is inhibited by an elevated salt concentration. The SSB mutant lacking SSB-Ct also inhibits RecO-mediated DNA annealing activity in a salt-dependent manner. Neither RecO nor RecOR dissociates SSB from ssDNA. Therefore, in E. coli, SSB recruits RMPs to ssDNA through SSB-Ct, and RMPs are likely to alter the conformation of SSB-bound ssDNA without SSB dissociation to initiate annealing or recombination. Intriguingly, Deinococcus radiodurans RecO does not bind SSB-Ct and weakly interacts with the peptide in the presence of RecR, suggesting the diverse mechanisms of DNA repair pathways mediated by RecO in different organisms.

RecF, together with RecO and RecR, belongs to a ubiquitous group of recombination mediators (RMs) that includes eukaryotic proteins such as Rad52 and BRCA2. RMs help maintain genome stability in the presence of DNA damage by loading RecA‐like recombinases and displacing single‐stranded DNA‐binding proteins. Here, we present the crystal structure of RecF from Deinococcus radiodurans. RecF exhibits a high degree of structural similarity with the head domain of Rad50, but lacks its long coiled‐coil region. The structural homology between RecF and Rad50 is extensive, encompassing the ATPase subdomain and the so‐called ‘Lobe II’ subdomain of Rad50. The pronounced structural conservation between bacterial RecF and evolutionarily diverged eukaryotic Rad50 implies a conserved mechanism of DNA binding and recognition of the boundaries of double‐stranded DNA regions. The RecF structure, mutagenesis of conserved motifs and ATP‐dependent dimerization of RecF are discussed with respect to its role in promoting presynaptic complex formation at DNA damage sites.

Calcium-independent phospholipase A2β (iPLA2β) regulates important physiological processes including inflammation, calcium homeostasis and apoptosis. It is genetically linked to neurodegenerative disorders including Parkinson’s disease. Despite its known enzymatic activity, the mechanisms underlying iPLA2β-induced pathologic phenotypes remain poorly understood. Here, we present a crystal structure of iPLA2β that significantly revises existing mechanistic models. The catalytic domains form a tight dimer. They are surrounded by ankyrin repeat domains that adopt an outwardly flared orientation, poised to interact with membrane proteins. The closely integrated active sites are positioned for cooperative activation and internal transacylation. The structure and additional solution studies suggest that both catalytic domains can be bound and allosterically inhibited by a single calmodulin. These features suggest mechanisms of iPLA2β cellular localization and activity regulation, providing a basis for inhibitor development. Furthermore, the structure provides a framework to investigate the role of neurodegenerative mutations and the function of iPLA2β in the brain.

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To date two classes of shikimate dehydrogenases have been identified and characterized, YdiB and AroE. YdiB is a bifunctional enzyme that catalyzes the reversible reductions of dehydroquinate to quinate and dehydroshikimate to shikimate in the presence of either NADH or NADPH. In contrast, AroE catalyzes the reversible reduction of dehydroshikimate to shikimate in the presence of NADPH. Here we report the crystal structure and biochemical characterization of HI0607, a novel class of shikimate dehydrogenase annotated as shikimate dehydrogenase-like. The kinetic properties of HI0607 are remarkably different from those of AroE and YdiB. In comparison with YdiB, HI0607 catalyzes the oxidation of shikimate but not quinate. The turnover rate for the oxidation of shikimate is ϳ1000-fold lower compared with that of AroE. Phylogenetic analysis reveals three independent clusters representing three classes of shikimate dehydrogenases, namely AroE, YdiB, and this newly characterized shikimate dehydrogenase-like protein. In addition, mutagenesis studies of two invariant residues, Asp-103 and Lys-67, indicate that they are important catalytic groups that may function as a catalytic pair in the shikimate dehydrogenase reaction. This is the first study that describes the crystal structure as well as mutagenesis and mechanistic analysis of this new class of shikimate dehydrogenase.The shikimate pathway occupies a central position for aromatic biosynthesis in microbes and plants but is not present in humans and other higher animals. The absence of the shikimate pathway in animals makes it an ideal target for herbicide and anti-microbial drug design. Recently the shikimate pathway was identified in apicomplexan parasites, including Toxoplasma gondii and Plasmodium falciparum, which has renewed interest in better understanding the enzymes in the pathway (1, 2). The importance of the shikimate pathway is exemplified by the common herbicide glyphosate, which inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase. Recent studies have also shown that glyphosate blocks the shikimate pathway in apicomplexan parasites and is effective in controlling their growth (3). The shikimate pathway consists of seven enzymatic steps initiated by the condensation of phosphoenolpyruvate and erythrose-4-phosphate by 3-deoxy-Darabino-heptolosonate 7-phosphate synthase. The last enzymatic step produces the branch point intermediate, chorismate, which serves in turn as the precursor for a number of pathways including those involved in aromatic amino acid, phytoalexin, flavanoid, and lignin biosynthesis.The fourth enzyme in the pathway, shikimate dehydrogenase (shikimate:NADP ϩ oxidoreductase; EC 1.1.1.25), is involved in the NADPH-dependent reduction of dehydroshikimate to shikimate. This enzymatic reaction proceeds in both the forward and reverse direction with similar rates and similar Michaelis constants for substrates in either direction. Kinetic studies with substrate analogues and isotope exchange demonstrated that the shikimate dehydrogena...

This paper describes the structural analysis of the native form of laccase from Trametes hirsuta at 1.8 A resolution. This structure provides a basis for the elucidation of the mechanism of catalytic action of these ubiquitous proteins. The 1.8 A resolution native structure provided a good level of structural detail compared with many previously reported laccase structures. A brief comparison with the active sites of other laccases is given.

Rate constants and activation parameters have been determined for the internal electron transfer from type 1 (T1) to type 3 (T3) copper ions in laccase from both the fungus Trametes hirsuta and the lacquer tree Rhus vernicifera, using the pulse radiolysis method. The rate constant at 298 K and the enthalpy and entropy of activation were 25 ± 1 s )1 , 39.7 ± 5.0 kJAEmol )1 and )87 ± 9 JAEmol )1 AEK )1 for the fungal enzyme and 1.1 ± 0.1 s )1 , 9.8 ± 0.2 kJAEmol )1 and )211 ± 3 JAEmol )1 AEK )1 for the tree enzyme. The initial reduction of the T1 site by pulse radiolytically produced CO À 2 radicals was direct in the case of T. hirsuta laccase, but occured indirectly via a disulfide radical in R. vernicifera. The equilibrium constant that characterizes the electron transfer from T1 to T3 copper ions was 0.4 for T. hirsuta laccase and 1.5 for R. vernicifera laccase, leading to full reduction of the T1 site occurring at 2.9 ± 0.2 electron equivalents for T. hirsuta and 4 electron equivalents for R. vernicifera laccase. These results were compared with each other and with those for the same process in other multicopper oxidases, ascorbate oxidase and Streptomyces coelicolor laccase, using available structural information and electron transfer theory. DatabaseProtein structure data for Trametes hirsuta laccase are available in the wwPDB database under the accession number 3FPX. Sequence references are Q94ID0 for Rhus vernicifera laccase and B2L9C1 for Trametes hirsuta laccase

Mycobacteria have two genetically distinct pathways for the homology-directed repair of DNA double-strand breaks: homologous recombination (HR) and single-strand annealing (SSA). HR is abolished by deletion of RecA and reduced in the absence of the AdnAB helicase/nuclease. By contrast, SSA is RecA-independent and requires RecBCD. Here we examine the function of RecO in mycobacterial DNA recombination and repair. Loss of RecO elicits hypersensitivity to DNA damaging agents similar to that caused by deletion of RecA. We show that RecO participates in RecA-dependent HR in a pathway parallel to the AdnAB pathway. We also find that RecO plays a role in the RecA-independent SSA pathway. The mycobacterial RecO protein displays a zinc-dependent DNA binding activity in vitro and accelerates the annealing of SSB-coated single-stranded DNA. These findings establish a role for RecO in two pathways of mycobacterial DNA double-strand break repair and suggest an in vivo function for the DNA annealing activity of RecO proteins, thereby underscoring their similarity to eukaryal Rad52.