A novel reduced flavin mononucleotide-dependent methanesulfonate sulfonatase encoded by the sulfur-regulated msu operon of Pseudomonas aeruginosa.
When Pseudomonas aeruginosa is grown with organosulfur compounds as sulfur sources, it synthesizes a set of proteins whose synthesis is repressed in the presence of sulfate, cysteine, or thiocyanate (so-called sulfate starvation-induced proteins). The gene encoding one of these proteins, PA13, was isolated from a cosmid library of P. aeruginosa PAO1 and sequenced. It encoded a 381-amino-acid protein that was related to several reduced flavin mononucleotide (FMNH2)-dependent monooxygenases, and it was the second in an operon of three genes, which we have named msuEDC. The MsuD protein catalyzed the desulfonation of alkanesulfonates, requiring oxygen and FMNH2 for the reaction, and showed highest activity with methanesulfonate. MsuE was an NADH-dependent flavin mononucleotide (FMN) reductase, which provided reduced FMN for the MsuD enzyme. Expression of the msu operon was analyzed with a transcriptional msuD::xylE fusion and was found to be repressed in the presence of sulfate, sulfite, sulfide, or cysteine and derepressed during growth with methionine or alkanesulfonates. Growth with methanesulfonate required an intact cysB gene, and the msu operon is therefore part of the cys regulon, since sulfite utilization was found to be CysB independent in this species. Measurements of msuD::xylE expression in cysN and cysI genetic backgrounds showed that sulfate, sulfite, and sulfide or cysteine play independent roles in negatively regulating msu expression, and sulfonate utilization therefore appears to be tightly regulated. (+info)
Localization of two phylloquinones, QK and QK', in an improved electron density map of photosystem I at 4-A resolution.
An improved electron density map of photosystem I from Synechococcus elongatus calculated at 4-A resolution for the first time reveals a second phylloquinone molecule and thereby completes the set of cofactors constituting the electron transfer system of this iron-sulfur type photosynthetic reaction center: six chlorophyll a, two phylloquinones, and three Fe4S4 clusters. The location of the newly identified phylloquinone pair, the individual plane orientations of these molecules, and the resulting distances to other cofactors of the electron transfer system are discussed and compared with those determined by magnetic resonance techniques. (+info)
Analysis of zinc binding sites in protein crystal structures.
The geometrical properties of zinc binding sites in a dataset of high quality protein crystal structures deposited in the Protein Data Bank have been examined to identify important differences between zinc sites that are directly involved in catalysis and those that play a structural role. Coordination angles in the zinc primary coordination sphere are compared with ideal values for each coordination geometry, and zinc coordination distances are compared with those in small zinc complexes from the Cambridge Structural Database as a guide of expected trends. We find that distances and angles in the primary coordination sphere are in general close to the expected (or ideal) values. Deviations occur primarily for oxygen coordinating atoms and are found to be mainly due to H-bonding of the oxygen coordinating ligand to protein residues, bidentate binding arrangements, and multi-zinc sites. We find that H-bonding of oxygen containing residues (or water) to zinc bound histidines is almost universal in our dataset and defines the elec-His-Zn motif. Analysis of the stereochemistry shows that carboxyl elec-His-Zn motifs are geometrically rigid, while water elec-His-Zn motifs show the most geometrical variation. As catalytic motifs have a higher proportion of carboxyl elec atoms than structural motifs, they provide a more rigid framework for zinc binding. This is understood biologically, as a small distortion in the zinc position in an enzyme can have serious consequences on the enzymatic reaction. We also analyze the sequence pattern of the zinc ligands and residues that provide elecs, and identify conserved hydrophobic residues in the endopeptidases that also appear to contribute to stabilizing the catalytic zinc site. A zinc binding template in protein crystal structures is derived from these observations. (+info)
The aconitase of yeast. IV. Studies on iron and sulfur in yeast aconitase.
Chemical analyses were carried out to determine the active components of the crystalline aconitase [EC 22.214.171.124] of Candida lipolytica. The enzyme contained 2 atoms of non-heme iron, 1 atom of labile sulfur, and 6 sulfhydryl groups per molecule. One atom of the non-heme iron was released by the addition of metal-chelating agents such as sodium citrate, sodium nitrilotriacetate (NTA) or sodium ethylenediaminetetraacetate (EDTA) without loss of the enzyme activity. The non-heme iron and labile sulfur were released by the addition of sulfhydryl reagents such as rho-chloromercuribenzoate (PCMB), sodium mersalyl or urea with loss of the enzyme activity. o-Phenanthroline reacted with the iron atoms in the enzyme at pH 6.0 with loss of the activity. These results show that yeast aconitase is an iron-sulfur protein and that only one of the two non-heme iron atoms is essential for enzyme activity. (+info)
Dense populations of a giant sulfur bacterium in Namibian shelf sediments.
A previously unknown giant sulfur bacterium is abundant in sediments underlying the oxygen minimum zone of the Benguela Current upwelling system. The bacterium has a spherical cell that exceeds by up to 100-fold the biovolume of the largest known prokaryotes. On the basis of 16S ribosomal DNA sequence data, these bacteria are closely related to the marine filamentous sulfur bacteria Thioploca, abundant in the upwelling area off Chile and Peru. Similar to Thioploca, the giant bacteria oxidize sulfide with nitrate that is accumulated to +info)
Kinetics and inhibition of recombinant human cystathionine gamma-lyase. Toward the rational control of transsulfuration.
The gene encoding human cystathionine gamma-lyase was cloned from total cellular Hep G2 RNA. Fusion to a T7 promoter allowed expression in Escherichia coli, representing the first mammalian cystathionine gamma-lyase overproduced in a bacterial system. About 90% of the heterologous gene product was insoluble, and renaturation experiments from purified inclusion bodies met with limited success. About 5 mg/liter culture of human cystathionine gamma-lyase could also be extracted from the soluble lysis fraction, employing a three-step native procedure. While the enzyme showed high gamma-lyase activity toward L-cystathionine (Km = 0.5 mM, Vmax = 2.5 units/mg) with an optimum pH of 8.2, no residual cystathionine beta-lyase behavior and only marginal reactivity toward L-cystine and L-cysteine were detected. Inhibition studies were performed with the mechanism-based inactivators propargylglycine, trifluoroalanine, and aminoethoxyvinylglycine. Propargylglycine inactivated human cystathionine gamma-lyase much more strongly than trifluoroalanine, in agreement with the enzyme's preference for C-gamma-S bonds. Aminoethoxyvinylglycine showed slow and tight binding characteristics with a Ki of 10.5 microM, comparable with its effect on cystathionine beta-lyase. The results have important implications for the design of specific inhibitors for transsulfuration components. (+info)
Role of XDHC in Molybdenum cofactor insertion into xanthine dehydrogenase of Rhodobacter capsulatus.
Rhodobacter capsulatus xanthine dehydrogenase (XDH) is composed of two subunits, XDHA and XDHB. Immediately downstream of xdhB, a third gene was identified, designated xdhC, which is cotranscribed with xdhAB. Interposon mutagenesis revealed that the xdhC gene product is required for XDH activity. However, XDHC is not a subunit of active XDH, which forms an alpha2beta2 heterotetramer in R. capsulatus. It was shown that XDHC neither is a transcriptional regulator for xdh gene expression nor influences XDH stability. To analyze the function of XDHC for XDH in R. capsulatus, inactive XDH was purified from an xdhC mutant strain. Analysis of the molybdenum cofactor content of this enzyme demonstrated that in the absence of XDHC, no molybdopterin cofactor MPT is present in the XDHAB tetramer. In contrast, absorption spectra of inactive XDH isolated from the xdhC mutant revealed the presence of iron-sulfur clusters and flavin adenine dinucleotide, demonstrating that XDHC is not required for the insertion of these cofactors. The absence of MPT from XDH isolated from an xdhC mutant indicates that XDHC either acts as a specific MPT insertase or might be a specific chaperone facilitating the insertion of MPT and/or folding of XDH during or after cofactor insertion. (+info)
Thiomicrospira kuenenii sp. nov. and Thiomicrospira frisia sp. nov., two mesophilic obligately chemolithoautotrophic sulfur-oxidizing bacteria isolated from an intertidal mud flat.
Two new members of the genus Thiomicrospira were isolated from an intertidal mud flat sample with thiosulfate as the electron donor and CO2 as carbon source. On the basis of differences in genotypic and phenotypic characteristics, it is proposed that strain JB-A1T (= DSM 12350T) and strain JB-A2T (= DSM 12351T) are members of two new species, Thiomicrospira kuenenii and Thiomicrospira frisia, respectively. The cells were Gram-negative vibrios or slightly bent rods. Strain JB-A1T was highly motile, whereas strain JB-A2T showed a much lower degree of motility combined with a strong tendency to form aggregates. Both organisms were obligately autotrophic and strictly aerobic. Nitrate was not used as electron acceptor. Chemolithoautotrophic growth was observed with thiosulfate, tetrathionate, sulfur and sulfide. Neither isolate was able to grow heterotrophically. For strain JB-A1T, growth was observed between pH values of 4.0 and 7.5 with an optimum at pH 6.0, whereas for strain JB-A2T, growth was observed between pH 4.2 and 8.5 with an optimum at pH 6.5. The temperature limits for growth were between 3.5 and 42 degrees C and 3.5 and 39 degrees C, respectively. The optimum growth temperature for strain JB-A1T was between 29 and 33.5 degrees C, whereas strain JB-A2T showed optimal growth between 32 and 35 degrees C. The mean maximum growth rate on thiosulfate was 0.35 h-1 for strain JB-A1T and 0.45 h-1 for strain JB-A2T. (+info)