MioC is an FMN-binding protein that is essential for Escherichia coli biotin synthase activity in vitro. (25/467)

Biotin synthase is required for the conversion of dethiobiotin to biotin and requires a number of accessory proteins and small molecule cofactors for activity in vitro. We have previously identified two of these proteins as flavodoxin and ferredoxin (flavodoxin) NADP(+) reductase. We now report the identification of MioC as a third essential protein, together with its cloning, purification, and characterization. Purified MioC has a UV-visible spectrum characteristic of a flavoprotein and contains flavin mononucleotide. The presence of flavin mononucleotide and the primary sequence similarity to flavodoxin suggest that MioC may function as an electron transport protein. The role of MioC in the biotin synthase reaction is discussed, and the structure and function of MioC is compared with that of flavodoxin.  (+info)

Characterization of two sulfurtransferase isozymes from Arabidopsis thaliana. (26/467)

Sulfurtransferases transfer a sulfane atom from a donor substrate to a thiophilic acceptor molecule. Recently a sulfurtransferase specific for the substrate 3-mercaptopyruvate was isolated from Arabidopsis thaliana [Papenbrock, J. & Schmidt, A. (2000) Eur. J. Biochem. 267, 145-154]. In this study a second sulfurtransferase from Arabidopsis was characterized and compared to the enzyme described previously. Sequences of the mature proteins had an identity of 77.7%. The plant sulfurtransferases formed a distinct group within the known eukaryotic sulfurtransferases. When Southern blots were hybridized with labelled cDNA fragments from each of the plant sulfurtransferases the same pattern of bands was obtained indicating the existence of only these two closely related sulfurtransferases. The new sulfurtransferase was expressed in Escherichia coli fused with an N-terminal His6-tag, purified and tested for enzyme activity. Like the first enzyme, the newly isolated protein preferred 3-mercaptopyruvate to thiosulfate as substrate. The Km of both enzymes determined for 3-mercaptopyruvate and cyanide were almost identical. As a result of database searches it became obvious that sulfurtransferase proteins from higher plants showed high similarities to small senescence- and stress-induced proteins. To prove the involvement of sulfurtransferases in senescence-associated processes 3-mercaptopyruvate sulfurtransferase activity was determined in crude protein extracts from Arabidopsis plants of different ages. 3-mercaptopyruvate sulfurtransferase activity and steady-state RNA levels of sulfurtransferases increased with increasing age. However, steady-state protein levels as measured by using an antibody against the sulfurtransferase protein expressed previously decreased. Putative roles of sulfurtransferases in senescence-associated processes are discussed.  (+info)

Plant mercaptopyruvate sulfurtransferases: molecular cloning, subcellular localization and enzymatic activities. (27/467)

Mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2) and thiosulfate sulfurtransferase (TST, rhodanese, EC 2.8.1.1) are evolutionarily related enzymes that catalyze the transfer of sulfur ions from mercaptopyruvate and thiosulfate, respectively, to cyanide ions. We have isolated and characterized two cDNAs, AtMST1 and AtMST2, that are Arabidopsis homologs of TST and MST from other organisms. Deduced amino-acid sequences showed similarity to each other, although MST1 has a N-terminal extension of 57 amino acids containing a targeting sequence. MST1 and MST2 are located in mitochondria and cytoplasm, respectively, as shown by immunoblot analysis of subcellular fractions and by green fluorescent protein (GFP) analysis. However, some regions of MST1 fused to GFP were found to target not only mitochondria, but also chloroplasts, suggesting that the regions on the targeting sequence recognized by protein import systems of mitochondria and chloroplasts are not identical. Recombinant proteins, expressed in Escherichia coli, exhibited MST/TST activity ratios determined from kcat/Km values of 11 and 26 for MST1 and MST2, respectively. This indicates that the proteins encoded by both AtMST1 and AtMST2 are MST rather than TST type. One of the hypotheses proposed so far for the physiological function of MST and TST concerns iron-sulfur cluster assembly. In order to address this possibility, a T-DNA insertion Arabidopsis mutant, in which the AtMST1 was disrupted, was isolated by PCR screening of T-DNA mutant libraries. However, the mutation had no effect on levels of iron-sulfur enzyme activities, suggesting that MST1 is not directly involved in iron-sulfur cluster assembly.  (+info)

Engineering hydrogen sulfide production and cadmium removal by expression of the thiosulfate reductase gene (phsABC) from Salmonella enterica serovar typhimurium in Escherichia coli. (28/467)

The thiosulfate reductase gene (phsABC) from Salmonella enterica serovar Typhimurium was expressed in Escherichia coli to overproduce hydrogen sulfide from thiosulfate for heavy metal removal (or precipitation). A 5.1-kb DNA fragment containing phsABC was inserted into the pMB1-based, high-copy, isopropyl-beta-D-thiogalactopyranoside-inducible expression vector pTrc99A and the RK2-based, medium-copy, m-toluate-inducible expression vector pJB866, resulting in plasmids pSB74 and pSB77. A 3. 7-kb DNA fragment, excluding putative promoter and regulatory regions, was inserted into the same vectors, making plasmids pSB103 and pSB107. E. coli DH5alpha strains harboring the phsABC constructs showed higher thiosulfate reductase activity and produced significantly more sulfide than the control strains under both aerobic and anaerobic conditions. Among the four phsABC constructs, E. coli DH5alpha (pSB74) produced thiosulfate reductase at the highest level and removed the most cadmium from solution under anaerobic conditions: 98% of all concentrations up to 150 microM and 91% of 200 microM. In contrast, a negative control did not produce any measurable sulfide and removed very little cadmium from solution. Energy-dispersive X-ray spectroscopy revealed that the metal removed from solution precipitated as a complex of cadmium and sulfur, most likely cadmium sulfide.  (+info)

A mutation in the gene for the neurotransmitter receptor-clustering protein gephyrin causes a novel form of molybdenum cofactor deficiency. (29/467)

Gephyrin was originally identified as a membrane-associated protein that is essential for the postsynaptic localization of receptors for the neurotransmitters glycine and GABA(A). A sequence comparison revealed homologies between gephyrin and proteins necessary for the biosynthesis of the universal molybdenum cofactor (MoCo). Because gephyrin expression can rescue a MoCo-deficient mutation in bacteria, plants, and a murine cell line, it became clear that gephyrin also plays a role in MoCo biosynthesis. Human MoCo deficiency is a fatal disease resulting in severe neurological damage and death in early childhood. Most patients harbor MOCS1 mutations, which prohibit formation of a precursor, or carry MOCS2 mutations, which abrogate precursor conversion to molybdopterin. The present report describes the identification of a gephyrin gene (GEPH) deletion in a patient with symptoms typical of MoCo deficiency. Biochemical studies of the patient's fibroblasts demonstrate that gephyrin catalyzes the insertion of molybdenum into molybdopterin and suggest that this novel form of MoCo deficiency might be curable by molybdate supplementation.  (+info)

Nuclear localization of yeast Nfs1p is required for cell survival. (30/467)

Saccharomyces cerevisiae Nfs1p is mainly found in the mitochondrial matrix and has been shown to participate in iron-sulfur cluster assembly. We show here that Nfs1p contains a potential nuclear localization signal, RRRPR, in its mature part. When this sequence was mutated to RRGSR, the mutant protein could not restore cell growth under chromosomal NFS1-depleted conditions. However, this mutation did not affect the function of Nfs1p in biogenesis of mitochondrial iron-sulfur proteins. The growth defect of the mutant was complemented by simultaneous expression of the mature Nfs1p, which contains the intact nuclear localization signal but lacks its mitochondrial-targeting presequence. These results suggest that a fraction of Nfs1p is localized in the nucleus and is essential for cell viability.  (+info)

The miaA mutator phenotype of Escherichia coli K-12 requires recombination functions. (31/467)

miaA mutants, which contain A-37 instead of the ms(2)i(6)A-37 hypermodification in their tRNA, show a moderate mutator phenotype leading to increased GC-->TA transversion. We show that the miaA mutator phenotype is dependent on recombination functions similar to, but not exactly the same as, those required for translation stress-induced mutagenesis.  (+info)

Tissue and intracellular distribution of rhodanese and mercaptopyruvate sulphurtransferase in ruminants and birds. (32/467)

Cyanide detoxification is catalysed by two enzymes: rhodanese [thiosulphate: cyanide sulphurtransferase, E.C. 2.8.1.1], and 3-mercaptopyruvate sulphurtransferase [3-MST, EC. 2.8.1.2]. In the present work, the activity of the two enzymes in the crude extracts of different tissues and in the mitochondrial and cytosolic fractions of tissues from some ruminants (camels, cattle and sheep) and birds (chickens and pigeons) have been compared. Rhodanese activity was almost exclusively present in the mitochondrial fraction. In ruminants and chickens the highest activity of rhodanese was found in the liver, followed by the kidney. In pigeons, however, the enzyme activity was the highest in the kidneys. In camels' tissues, the rhodanese activity was significantly (P < 0.05) lower than in cattle or sheep, and the enzyme activities in the two latter species were similar. The activity of 3-MST in the crude extract of tissues from camels was similar to that in sheep, but higher than that in cattle. The enzyme activity was equally distributed between the mitochondrial and cytosolic fractions in the liver and kidneys of camels, cattle and sheep.  (+info)