Twin-arginine Translocation Signal Sequence Research Articles

Production and characterisation of a novel actinobacterial DyP-type peroxidase and its application in coupling of phenolic monomers

The extracellular peroxidase from Streptomyces albidoflavus BSII#1 was purified to near homogeneity using sequential steps of acid and acetone precipitation, followed by ultrafiltration. The purified peroxidase was characterised and tested for the ability to catalyse coupling reactions between selected phenolic monomer pairs. A 46-fold purification of the peroxidase was achieved, and it was shown to be a 46 kDa haem peroxidase. Unlike other actinobacteria-derived peroxidases, it was only inhibited (27 % inhibition) by relatively high concentrations of sodium azide (5 mM) and was capable of oxidising eleven (2,4-dichlorophenol, 2,6-dimethoxyphenol, 4-tert-butylcatechol, ABTS, caffeic acid, catechol, guaiacol, l-DOPA, o-aminophenol, phenol, pyrogallol) of the seventeen substrates tested. The peroxidase remained stable at temperatures of up to 80 °C for 60 min and retained >50 % activity after 24 h between pH 5.0–9.0, but was most sensitive to incubation with hydrogen peroxide (H2O2; 0.01 mM), l-cysteine (0.02 mM) and ascorbate (0.05 mM) for one hour. It was significantly inhibited by all organic solvents tested (p ≤ 0.05). The Km and Vmax values of the partially purified peroxidase with the substrate 2,4-DCP were 0.95 mM and 0.12 mmol min−1, respectively. The dyes reactive blue 4, reactive black 5, and Azure B, were all decolourised to a certain extent: approximately 30 % decolourisation was observed after 24 h (1 μM dye). The peroxidase successfully catalysed coupling reactions between several phenolic monomer pairs including catechin-caffeic acid, catechin-catechol, catechin-guaiacol and guaiacol-syringaldazine under the non-optimised conditions used in this study. Genome sequencing confirmed the identity of strain BSII#1 as a S. albidoflavus strain. In addition, the genome sequence revealed the presence of one peroxidase gene that includes the twin arginine translocation signal sequence of extracellular proteins. Functional studies confirmed that the peroxidase produced by S. albidoflavus BSII#1 is part of the dye-decolourising peroxidase (DyP-type) family.

Cross-Species Functional Conservation and Possible Origin of the N-Terminal Specificity Domain of Mitochondrial Presequences.

Plants have two endosymbiotic organelles, chloroplast and mitochondrion. Although they have their own genomes, proteome assembly in these organelles depends on the import of proteins encoded by the nuclear genome. Previously, we elucidated the general design principles of chloroplast and mitochondrial targeting signals, transit peptide, and presequence, respectively, which are highly diverse in primary structure. Both targeting signals are composed of N-terminal specificity domain and C-terminal translocation domain. Especially, the N-terminal specificity domain of mitochondrial presequences contains multiple arginine residues and hydrophobic sequence motif. In this study we investigated whether the design principles of plant mitochondrial presequences can be applied to those in other eukaryotic species. We provide evidence that both presequences and import mechanisms are remarkably conserved throughout the species. In addition, we present evidence that the N-terminal specificity domain of presequence might have evolved from the bacterial TAT (twin-arginine translocation) signal sequence.

Novel reductive dehalogenases from the marine sponge associated bacterium Desulfoluna spongiiphila.

Desulfoluna spongiiphila strain AA1 is an organohalide respiring bacterium, isolated from the marine sponge Aplysina aerophoba, that can use brominated and iodinated phenols, in addition to sulfate and thiosulfate as terminal electron acceptors. The genome of Desulfoluna spongiiphila strain AA1 is approximately 6.5 Mb. Three putative reductive dehalogenase (rdhA) genes involved in respiratory metabolism of organohalides were identified within the sequence. Conserved motifs found in respiratory reductive dehalogenases (a twin arginine translocation signal sequence and two iron-sulfur clusters) were present in all three putative AA1 rdhA genes. Transcription of one of the three rdhA genes was significantly upregulated during respiration of 2,6-dibromophenol and sponge extracts. Strain AA1 appears to have the ability to synthesize cobalamin, the key cofactor of most characterized reductive dehalogenase enzymes. The genome contains genes involved in cobalamin synthesis and uptake and can grow without cobalamin supplementation. Identification of this target gene associated with debromination lays the foundation for understanding how dehalogenating bacteria control the fate of organohalide compounds in sponges and their role in a symbiotic organobromine cycle. Inthe sponge environment, D. spongiiphila strain AA1 may thus take advantage of both brominated compounds and sulfate as electron acceptors for respiration.

Heterologously expressed arsenite oxidase: A system to study biogenesis and structure/function relationships of the enzyme family

Studies of native arsenite oxidases from Ralstonia sp. S22 and Rhizobium sp. NT-26 raised two major questions. The first one concerns the mode of the enzyme's membrane-association. It has been suggested that a hypothetical not conserved protein could account for this variable association. Expression of the wild type arsenite oxidase in Escherichia coli allowed us to study the cellular localization of this enzyme in the absence of such a hypothetical partner. The results with the Ralstonia sp. S22 enzyme suggest that no additional protein is required for membrane association. The second question addresses the influence of the disulfide bridge in the small Rieske subunit, conspicuously absent in the Rhizobium sp. NT-26 enzyme, on the properties of the [2Fe–2S] center. The disulfide bridge is considered to be formed only after translocation of the enzyme to the periplasm. To address this question we thus first expressed the enzyme in the absence of its Twin-arginine translocation signal sequence. The spectral and redox properties of the cytoplasmic enzyme are unchanged compared to the periplasmic one. We finally studied a disulfide bridge mutant, Cys106Ala, devoid of the first Cys involved in the disulfide bridge formation. This mutation, proposed to have a strong effect on redox and catalytic properties of the Rieske protein in Rieske/cytb complexes, had no significant effect on properties of the Rieske protein from arsenite oxidase. Our present results demonstrate that the effects attributed to the disulfide bridge in the Rieske/cytb complexes are likely to be secondary effects due to conformational changes.

Functional characterization of the trigger factor protein PceT of tetrachloroethene-dechlorinating Desulfitobacterium hafniense Y51

Desulfitobacterium hafniense strain Y51 dechlorinates tetrachloroethene to cis-1,2-dichloroethene (cis-DCE) via trichloroethene by the action of the PceA reductive dehalogenase encoded by pceA. The pceA gene constitutes a gene cluster with pceB, pceC, and pceT. However, the gene components, except for pceA, still remained to be characterized. In the present study, we characterized the function of PceT. PceT of strain Y51 showed a sequence homology with trigger factor proteins, although it is evolutionally distant from the well-characterized trigger factor protein of Escherichia coli. The PceT protein tagged with 6x histidine was expressed as a soluble form in E. coli. The recombinant PceT fusion protein exhibited peptidyl-proryl cis-trans isomerase activity toward the chromogenic peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The PceT fusion protein also exhibited chaperon activity towards the chemically denatured citrate synthase. Immunoprecipitation analysis using antibodies raised against PceA and PceT demonstrated that PceT specifically binds to the precursor form of PceA with an N-terminal twin-arginine translocation (TAT) signal sequence. On the other hand, PceT failed to bind the mature form of PceA that lost the TAT signal sequence. This is the first report in dehalorespiring bacteria, indicating that PceT is responsible for the correct folding of the precursor PceA.

Characterization of the Twin-Arginine Translocase Secretion System of Mycobacterium smegmatis

The twin-arginine translocation (TAT) system secretes fully folded proteins that contain a twin-arginine motif within their signal sequence across the cytoplasmic membrane in bacteria. Using a green fluorescent protein fused with a TAT signal sequence, we demonstrated that Mycobacterium smegmatis contains a TAT system. By inactivating individual genes, we showed that three genes (tatA, tatB, and tatC) are required for a functional TAT system in M. smegmatis. The tat mutants exhibited a decreased growth rate and altered colony morphology compared to the parent strain. Comparison of the secreted proteins of the deltatatC and parent strain by two-dimensional polyacrylamide gel electrophoresis revealed an alteration in the secretion of at least five proteins, and one of the major TAT-dependent secreted proteins was identified as beta-lactamase (BlaS). The genome of M. smegmatis was analyzed with the TATFIND program, and 49 putative TAT substrates were identified, including the succinate transporter DctP. Because disruption of the TAT secretion system has a direct effect on the physiology of M. smegmatis and homologs of the TAT proteins are also present in the genome of Mycobacterium tuberculosis, the TAT secretion system or its substrates may be good candidates for drug or vaccine development.