Article

Sulfide:quinone oxidoreductase from echiuran worm Urechis unicinctus.

Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao, 266003, China.
Marine Biotechnology (Impact Factor: 3.15). 04/2010; 13(1):93-107. DOI: 10.1007/s10126-010-9273-3
Source: PubMed

ABSTRACT Sulfide is a natural, widely distributed, poisonous substance, and sulfide:quinone oxidoreductase (SQR) has been identified to be responsible for the initial oxidation of sulfide in mitochondria. In this study, full-length SQR cDNA was cloned from the echiuran worm Urechis unicinctus, a benthic organism living in marine sediments. The protein consisted of 451 amino acids with a theoretical pI of 8.98 and molecular weight of 50.5 kDa. Subsequently, the SQR mRNA expression in different tissues was assessed by real-time reverse transcription and polymerase chain reaction and showed that the highest expression was in midgut, followed by anal sacs and coelomic fluid cells, and then body wall and hindgut. Furthermore, activated SQR was obtained by dilution refolding of recombinant SQR expression in E. coli, and the refolded product showed optimal activity at 37 °C and pH 8.5 and K (m) for ubiquinone and sulfide at 15.6 µM and 40.3 µM, respectively. EDTA and GSH had an activating effect on refolded SQR, while Zn(2+) caused decreased activity. Western blot showed that SQR in vivo was located in mitochondria and was ∼ 10 kDa heavier than the recombinant protein. In addition, SQR, detected by immunohistochemistry, was mainly located in the epithelium of all tissues examined. Ultrastructural observations of these tissues' epithelium by transmission electron microscopy provided indirect cytological evidence for its mitochondrial location. Interesting aspects of the U. unicinctus SQR amino acid sequence, its catalytic mechanism, and the different roles of these tissues in sulfide metabolic adaptation are also discussed.

0 Bookmarks
 · 
74 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Although the biogeochemistry of the two environmentally hazardous compounds arsenic and sulfide has been extensively investigated, the biological interference of these two toxic, but potentially energy-rich compounds has only been hypothesized and indirectly proven. Here we provide direct evidence for the first time that in the photosynthetic model organism Synechocystis sp. PCC6803 the two metabolic pathways are linked by co-regulated genes that are involved in arsenic transport and sulfide oxidation, and probably in sulfide based alternative photosynthesis. Although Synechocystis sp. PCC6803 is an obligate photoautotrophic cyanobacterium that grows via oxygenic photosynthesis we discovered that specific genes are activated in the presence of sulfide or arsenite to exploit the energy potentials of these chemicals. These genes form an operon that we termed suoRSCT, located on a transposable element of type IS4 on the plasmid pSYSM of the cyanobacterium. SuoS (sll5036) encodes a light dependent, type I sulfide:quinone oxidoreductase. The suoR (sll5035) gene downstream of suoS encodes a regulatory protein that belongs to the ArsR-type repressors that are normally involved in arsenic resistance. We found that this repressor has dual specificity, resulting in 200 fold induction of the operon upon either arsenite or sulfide exposure. The suoT gene encodes a transmembrane protein similar to chromate transporters but in fact functioning as arsenite importer at permissive concentrations. We propose that the proteins encoded in the suoRSCT operon might have played an important role under anaerobic, reducing conditions on primordial Earth and it was acquired by the cyanobacterium via horizontal gene transfer.
    Journal of bacteriology. 07/2014;
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 17β-hydroxysteroid dehydrogenases (17β-HSDs) are important enzymes catalyzing steroids biosynthesis and metabolism in vertebrates. Although studies indicate steroids play a potential role in reproduction of molluscs, little is known about the presence and function of 17β-HSDs in molluscs. In the present study, a full-length cDNA encoding 17β-HSD type 8 (17β-HSD8) was identified in the Zhikong scallop Chlamys farreri, which is 1,104bp in length with an open reading frame of 759bp encoding a protein of 252 amino acids. Phylogenetic analysis revealed that the C. farreri 17β-HSD8 (Cf-17β-HSD8) belongs to the short chain dehydrogenase/reductase family (SDR) and shares high homology with other 17β-HSD8 homologues. Catalytic activity assay in vitro demonstrated that the refolded Cf-17β-HSD8 expressed in E. coli could effectively convert estradiol-17β (E2) to estrone (E1), and weakly catalyze the conversion of testosterone (T) to androstenedione (A) in the presence of NAD(+). The Cf-17β-HSD8 mRNA was ubiquitously expressed in all tissues analyzed, including gonads. The expression levels of Cf-17β-HSD8 mRNA and protein increased with gametogenesis in both ovary and testis, and were significantly higher in testis than in ovary at growing stage and mature stage. Moreover, results of in situ hybridization and immunohistochemistry revealed that the mRNA and protein of Cf-17β-HSD8 were expressed in follicle cells and gametes at all stages except spermatozoa. Our findings suggest that Cf-17β-HSD8 may play an important role in regulating gametogenesis through modulating E2 levels in gonad of C. farreri.
    The Journal of steroid biochemistry and molecular biology 01/2014; · 3.98 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Sulfide is a common toxin to animals and is abundant in coastal and aquatic sediments. Sulfur dioxygenase (SDO) is thought to be the key enzyme involved in sulfide oxidation in some organisms. The echiuran worm, Urechis unicinctus, inhabits coastal sediment and tolerates high concentrations of sulfide. The SDO is presumably important for sulfide tolerance in U. unicinctus. The full-length cDNA of SDO from the echiuran worm U. unicinctus, proven to be located in the mitochondria, was cloned and the analysis of its sequence suggests that it belongs to the metallo-β-lactamase superfamily. The enzyme was produced using an E. coli expression system and the measured activity is approximately 0.80 U mg protein(-1). Furthermore, the expression of four sub-segments of the U. unicinctus SDO was accomplished leading to preliminary identification of functional domains of the enzyme. The identification of the conserved metal I (H113, H115, H169 and D188), metal II (D117, H118, H169 and H229) as well as the potential glutathione (GSH) (R197, Y231, M279 and I283) binding sites was determined by enzyme activity and GSH affinity measurements. The key residues responsible for SDO activity were identified by analysis of simultaneous mutations of residues D117 and H118 located close to the metal II binding site. The recombinant SDO from U. unicinctus was produced, purified and characterized. The metal binding sites in the SDO were identified and Y231 recognized as the mostly important amino acid residue for GSH binding. Our results show that SDO is located in the mitochondria where it plays an important role in sulfide detoxification of U. unicinctus.
    PLoS ONE 01/2013; 8(12):e81885. · 3.53 Impact Factor

Full-text

Download
3 Downloads
Available from
Aug 1, 2014

Similar Publications