The oxidized "as isolated" form of Paracoccus pantotrophus cytochrome cd1 nitrite reductase has a bis-histidinyl coordinated c heme and a histidine/tyrosine coordinated d1 heme. This form of the enzyme has previously been shown to be kinetically incompetent. Upon reduction, the coordination of both hemes changes and the enzyme is kinetically activated. Here, we show that P. pantotrophus NapC, a tetraheme c-type cytochrome belonging to a large family of such proteins, is capable of reducing, and hence activating, "as isolated" cytochrome cd1. NapC is the first protein from P. pantotrophus identified as being capable of this activation step and, given the periplasmic co-location and co-expression of the two proteins, is a strong candidate to be a physiological activation partner. (+info)
Dissimilation of cysteate via 3-sulfolactate sulfo-lyase and a sulfate exporter in Paracoccus pantotrophus NKNCYSA.
(2/19)
Paracoccus pantotrophus NKNCYSA utilizes (R)-cysteate (2-amino-3-sulfopropionate) as a sole source of carbon and energy for growth, with either nitrate or molecular oxygen as terminal electron acceptor, and the specific utilization rate of cysteate is about 2 mkat (kg protein)(-1). The initial degradative reaction is catalysed by an (R)-cysteate : 2-oxoglutarate aminotransferase, which yields 3-sulfopyruvate. The latter was reduced to 3-sulfolactate by an NAD-linked sulfolactate dehydrogenase [3.3 mkat (kg protein)(-1)]. The inducible desulfonation reaction was not detected initially in cell extracts. However, a strongly induced protein with subunits of 8 kDa (alpha) and 42 kDa (beta) was found and purified. The corresponding genes had similarities to those encoding altronate dehydratases, which often require iron for activity. The purified enzyme could then be shown to convert 3-sulfolactate to sulfite and pyruvate and it was termed sulfolactate sulfo-lyase (Suy). A high level of sulfite dehydrogenase was also induced during growth with cysteate, and the organism excreted sulfate. A putative regulator, OrfR, was encoded upstream of suyAB on the reverse strand. Downstream of suyAB was suyZ, which was cotranscribed with suyB. The gene, an allele of tauZ, encoded a putative membrane protein with transmembrane helices (COG2855), and is a candidate to encode the sulfate exporter needed to maintain homeostasis during desulfonation. suyAB-like genes are widespread in sequenced genomes and environmental samples where, in contrast to the current annotation, several presumably encode the desulfonation of 3-sulfolactate, a component of bacterial spores. (+info)
Y25S variant of Paracoccus pantotrophus cytochrome cd1 provides insight into anion binding by d1 heme and a rare example of a critical difference between solution and crystal structures.
(3/19)
Tyr25 is a ligand to the active site d1 heme in as isolated, oxidized cytochrome cd1 nitrite reductase from Paracoccus pantotrophus. This form of the enzyme requires reductive activation, a process that involves not only displacement of Tyr25 from the d1 heme but also switching of the ligands at the c heme from bis-histidinyl to His/Met. A Y25S variant retains this bis-histidinyl coordination in the crystal of the oxidized state that has sulfate bound to the d1 heme iron. This Y25S form of the enzyme does not require reductive activation, an observation previously interpreted as meaning that the presence of the phenolate oxygen of Tyr25 is the critical determinant of the requirement for activation. This interpretation now needs re-evaluation because, unexpectedly, the oxidized as prepared Y25S protein, unlike the wild type, has different heme iron ligands in solution at room temperature, as judged by magnetic circular dichroism and electron spin resonance spectroscopies, than in the crystal. In addition, the binding of nitrite and cyanide to oxidized Y25S cytochrome cd1 is markedly different from the wild type enzyme, thus providing insight into the affinity of the oxidized d1 heme ring for anions in the absence of the steric barrier presented by Tyr25. (+info)
Activation and catalysis of the di-heme cytochrome c peroxidase from Paracoccus pantotrophus.
(4/19)
Bacterial cytochrome c peroxidases contain an electron transferring (E) heme domain and a peroxidatic (P) heme domain. All but one of these enzymes are isolated in an inactive oxidized state and require reduction of the E heme by a small redox donor protein in order to activate the P heme. Here we present the structures of the inactive oxidized and active mixed valence enzyme from Paracoccus pantotrophus. Chain flexibility in the former, as expressed by the crystallographic temperature factors, is strikingly distributed in certain loop regions, and these coincide with the regions of conformational change that occur in forming the active mixed valence enzyme. On the basis of these changes, we postulate a series of events that occur to link the trigger of the electron entering the E heme from either pseudoazurin or cytochrome c(550) and the dissociation of a coordinating histidine at the P heme, which allows substrate access. (+info)
SoxV transfers electrons to the periplasm of Paracoccus pantotrophus - an essential reaction for chemotrophic sulfur oxidation.
(5/19)
The soxVW genes are located upstream of the sox gene cluster encoding the sulfur-oxidizing ability of Paracoccus pantotrophus. SoxV is highly homologous to CcdA, which is involved in cytochrome c maturation of P. pantotrophus. SoxV was shown to function in reduction of the periplasmic SoxW, which shows a CysXaaXaaCys motif characteristic for thioredoxins. From strain GBOmegaV, which carries an Omega-kanamycin-resistance-encoding interposon in soxV, and complementation analysis it was evident that SoxV but not the periplasmic SoxW was essential for lithoautotrophic growth of P. pantotrophus with thiosulfate. However, the thiosulfate-oxidizing activities of cell extracts from the wild-type and from strain GBOmegaV were similar, demonstrating that the low thiosulfate-oxidizing activity of strain GBOmegaV in vivo was not due to a defect in biosynthesis or maturation of proteins of the Sox system and suggesting that SoxV is part of a regulatory or catalytic system of the Sox system. Analysis of DNA sequences available from different organisms harbouring a Sox system revealed that soxVW genes are exclusively present in sox operons harbouring the soxCD genes, encoding sulfur dehydrogenase, suggesting that SoxCD might be a redox partner of SoxV. No complementation of the ccdA mutant P. pantotrophus TP43 defective in cytochrome c maturation was achieved by expression of soxV in trans, demonstrating that the high identity of SoxV and CcdA does not correspond to functional homology. (+info)
Identification of a transposable genomic island of Paracoccus pantotrophus DSM 11072 by its transposition to a novel entrapment vector pMMB2.
(6/19)
A novel shuttle entrapment vector, pMMB2, was used to identify a large transposable element, TnPpa1 (44.3 kb), of Paracoccus pantotrophus DSM 11072. TnPpa1 has a composite structure with divergently oriented copies of a cryptic transposon, Tn3434 (Tn3 family), located at both termini. The core region of the element contains a large set of putative genes, whose products show similarity to enzymes involved in central intermediary metabolism (e.g. tricarboxylic acid cycle or 2-methylcitrate cycle), transporters, transcriptional regulators and conserved proteins of unknown function. A 4.2 kb DNA segment of TnPpa1 is homologous to a region of chromosome cII of Rhodobacter sphaeroides 2.4.1, which exemplifies the mosaic structure of this element. TnPpa1 is bordered by 5 bp long directly repeated sequences and is located within a mega-sized replicon, pWKS5, in the DSM 11072 genome. Spontaneous inversion of the core region of TnPpa1 was detected in the host genome. Analysis of the distribution of TnPpa1 in three other strains of P. pantotrophus revealed that this element was present exclusively within DSM 11072, which suggests its relatively recent acquisition by lateral transfer. The identification of TnPpa1 (which may be considered a transposable genomic island) provides evidence for the transposition and lateral transfer of large DNA segments of chromosomal origin (carrying various housekeeping genes), which may have a great impact on the evolution of bacterial genomes. (+info)
Redefining Paracoccus denitrificans and Paracoccus pantotrophus and the case for a reassessment of the strains held by international culture collections.
(7/19)
An outline of the current taxonomic diversity of the genus Paracoccus is presented. A definitive summary is given of the valid type strains of Paracoccus denitrificans and Paracoccus pantotrophus and of culture collection strains that can be assigned to these species. The case is established for a critical reassessment of the P. denitrificans strains held by international culture collections, to ensure that they are assigned to the correct species. (+info)
The periplasmic thioredoxin SoxS plays a key role in activation in vivo of chemotrophic sulfur oxidation of Paracoccus pantotrophus.
(8/19)
The significance of the soxS gene product on chemotrophic sulfur oxidation of Paracoccus pantotrophus was investigated. The thioredoxin SoxS was purified, and the N-terminal amino acid sequence identified SoxS as the soxS gene product. The wild-type formed thiosulfate-oxidizing activity and Sox proteins during mixotrophic growth with succinate plus thiosulfate, while there was no activity, and only traces of Sox proteins, under heterotrophic conditions. The homogenote mutant strain GBOmegaS is unable to express the soxSR genes, of which soxR encodes a transcriptional regulator. Strain GBOmegaS cultivated mixotrophically showed about 22 % of the specific thiosulfate-dependent O(2) uptake rate of the wild-type, and when cultivated heterotrophically it produced 35 % activity. However, under both mixotrophic and heterotrophic conditions, strain GBOmegaS formed Sox proteins essential for sulfur oxidation in vitro at the same high level as the wild-type produced them during mixotrophic growth. Genetic complementation of strain GBOmegaS with soxS restored the activity upon mixotrophic and heterotrophic growth. Chemical complementation by reductants such as L-cysteine, DTT and tris(2-carboxyethyl)phosphine also restored the activity of strain GBOmegaS in the presence of chloramphenicol, which is an inhibitor of de novo protein synthesis. The data demonstrate that SoxS plays a key role in activation of the Sox enzyme system, and this suggests that SoxS is part of a novel type of redox control in P. pantotrophus. (+info)