Post-translational modifications of Desulfovibrio vulgaris Hildenborough sulfate reduction pathway proteins.

Recent developments in shotgun proteomics have enabled high-throughput studies of a variety of microorganisms at a proteome level and provide experimental validation for predicted open reading frames in the corresponding genome. More importantly, advances in mass spectrometric data analysis now allow mining of large proteomics data sets for the presence of post-translational modifications (PTMs). Although PTMs are a critical aspect of cellular activity, such information eludes cell-wide studies conducted at the transcript level. Here, we analyze several mass spectrometric data sets acquired using two-dimensional liquid chromatography tandem mass spectrometry, 2D-LC/MS/MS, for the sulfate reducing bacterium, Desulfovibrio vulgaris Hildenborough. Our searches of the raw spectra led us to discover several post-translationally modified peptides in D. vulgaris. Of these, several peptides containing a lysine with a +42 Da modification were found reproducibly across all data sets. Both acetylation and trimethylation have the same nominal +42 Da mass, and are therefore candidates for this modification. Several spectra were identified having markers for trimethylation, while one is consistent with an acetylation. Surprisingly, these modified peptides predominantly mapped to proteins involved in sulfate respiration. Other highly expressed proteins in D. vulgaris, such as enzymes involved in electron transport and other central metabolic processes, did not contain this modification. Decoy database searches were used to control for random spectrum/sequence matches. Additional validation for these modifications was provided by alternate workflows, for example, two-dimensional gel electrophoresis followed by mass spectrometry analysis of the dissimilatory sulfite reductase gamma-subunit (DsrC) protein. MS data for DsrC in this alternate workflow also contained the +42 Da modification at the same loci. Furthermore, the DsrC homologue in another sulfate reducing bacterium, Desulfovibrio desulfuricans G20, also showed similar +42 Da modifications in the same pathway. Here, we discuss our methods and implications of potential trimethylation in the D. vulgaris sulfate reduction pathway.

Tissue distribution and ontogeny of sulfotransferase enzymes in mice.

Sulfotransferases (Sults) are phase-II conjugation enzymes that catalyze the transfer of a sulfonate group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to target endo and xenobiotics. PAPS is formed from inorganic sulfate by the action of the enzyme PAPS synthase (PAPSs). In the present study, the tissue distribution and developmental changes in the mRNA expression of 11 Sult isozymes and 2 PAPSs isoforms in mice were quantified. Sult1a1, 1b1, 1c1, 1c2, 1d1, 1e1, 2a1/2, 2b1, 3a1, 4a1, 5a1, PAPSs1, and PAPSs2 mRNA expression was quantified in 14 tissues from male and female mice using the branched DNA signal amplification assay. Sult2a1/2 and 3a1 expression were highest in liver; Sult1b1, 2b1, and PAPSs2 in small intestine; Sult1a1 in large intestine; Sult1c2 in stomach; Sult1d1 in kidney; Sult1e1 in placenta; and Sult4a1 in brain. Sult1c1, 5a1, and PAPSs1 were ubiquitously expressed in most tissues. These enzymes demonstrated three different ontogenic expression patterns in liver. Sult1a1, 1c2, 1d1, 2a1/2, and PAPSs2 hepatic expression gradually increased from birth until about 3 weeks of age and then declined somewhat thereafter, Sult1c1 expression was highest before birth and declined after that, and Sult3a1 mRNA expression was very low in fetal livers and remained low until 30 days of age, when expression in females dramatically increased, whereas it never increased in males. The organ-specific distribution of Sults as well as the different expression of the Sults in young animals may affect the pharmacokinetic behavior and organ-specific toxicity of xenobiotics.

Novel bioluminescent assay of alkaline phosphatase using adenosine-3'-phosphate-5'-phosphosulfate as substrate and the luciferin-luciferase reaction and its application.

This paper describes a novel bioluminescent assay of alkaline phosphatase (ALP) utilizing ATP-sulfurylase and the luciferin-luciferase reaction. The principle governing the assay is as follows. Adenosine-3'-phosphate-5'-phosphosulfate, which serves as the substrate for ALP, is hydrolyzed enzymatically to produce adenosine-5'-phosphosulfate (APS). APS is converted into ATP by ATP-sulfurylase in the presence of pyrophosphate. The ATP produced is detected by the luciferin-luciferase reaction. The measurable range was 1 zmol to 100 fmol/assay and the detection limit at blank+3 SD was 10 zmol/assay. The coefficient of variation (CV, n=5) was examined at each point of the standard curve; the mean CV percentage was 4.47% (n=6). This assay system was applied to enzyme immunoassay of human chorionic gonadotropin and allele-specific PCR enzyme-linked immunosorbent assay of verotoxin gene using ALP as the label enzyme; 10(-2) mIU/mL hCG in urine and 5 pg of Escherichia coli O157 DNA could be assayed directly and with high sensitivity by the proposed method.

Development of a homologous transformation system for the opportunistic human pathogen Aspergillus fumigatus based on the sC gene encoding ATP sulfurylase.

The development of a homologous transformation system for the opportunistic human pathogenic fungus Aspergillus fumigatus is described. The system is based on the sC gene encoding ATP sulfurylase. Several A. fumigatus sC mutant strains were readily isolated by strong selection for selenate resistance. The coding region plus upstream and downstream regulatory sequences of the A. fumigatus sC gene were cloned by inverse PCR and then sequenced. Sequencing of the sC cDNA revealed the presence of five introns located within the first half of the gene. The A. fumigatus sC gene encodes a protein of 574 amino acids which is highly similar to ATP sulfurylases from the filamentous fungal species Aspergillus nidulans, Aspergillus terreus and Penicillium chrysogenum. By contrast, ATP sulfurylases from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe lack the C-terminal adenosine-5'-phosphosulfate kinase-like domain present in the filamentous fungal orthologues. A 3.8-kb DNA fragment amplified by PCR and containing the sC gene plus 5' and 3' flanking regions was cloned into pUC19 to give the vector pSCFUM. Transformation of two different sC mutant isolates with the plasmid pSCFUM established the functionality of this new homologous transformation system. Molecular analysis of sC+ transformants showed that up to 44% of transformed clones contained one or more copies of the entire plasmid integrated at the sC locus. This result also demonstrates the utility of the sC marker for targeting specific genetic constructs to the A. fumigatus sC locus, facilitating studies of gene regulation and function.

Real-time detection and quantification of adenosine triphosphate sulfurylase activity by a bioluminometric approach.

A real-time, sensitive, and simple assay for detection and quantification of adenosine triphosphate sulfurylase (ATP:sulfate adenylytransferase, EC 2.7.7.4) activity has been developed. The method is based on detection of ATP generated in the ATP sulfurylase reaction between APS and PPi by the firefly luciferase system. For the Saccharomyces cerevisiae ATP sulfurylase, the concentrations of APS and PPi at the half-maximal rate were found to be about 0.5 and 7 microM, respectively. The assay is sensitive and yields linear response between 0.1 microU and 50 mU. The method can be used for monitoring and quantification of recombinant ATP sulfurylase activity in Escherichia coli lysate, as well as for detection of the activity during different purification procedures.

Dissimilatory ATP sulfurylase from the hyperthermophilic sulfate reducer Archaeoglobus fulgidus belongs to the group of homo-oligomeric ATP sulfurylases.

In the hyperthermophilic sulfate reducer Archaeoglobus fulgidus DSM 4304T, two open reading frames (sat and ORF2) are located upstream of the aprBA genes encoding adenosine-5'-phosphosulfate (APS) reductase. sat-ORF2-aprBA probably form a transcriptional unit, since sat is preceded by putative promoter sequences and termination signals are found downstream of aprA. While the 117-residue ORF2 product does not show significant similarity to known proteins, the 456-residue, 52.78-kDa, sat-encoded polypeptide exhibits similarity to the homo-oligomeric adenosine triphosphate (ATP) sulfurylases from sulfur-oxidizing bacteria and from sulfate-assimilating bacteria and eukaryotes. Functional overexpression of sat in Escherichia coli proved that the encoded protein acts as an ATP sulfurylase. The recombinant protein was purified to homogeneity and found to be a homo-dimer. Comparison of sulfate and thiosulfate grown A. fulgidus revealed that ATP sulfurylase and APS reductase are constitutive enzymes. Distance matrix analyses allowed insights into the evolution of prokaryotic ATP sulfurylases.

Radioactive-electrophoretic assay of adenosine 5'-triphosphate sulfurylase activity in crude extracts with sulfate or selenate as a substrate.

An assay method for ATP sulfurylase is presented which employs Na2(35)SO4 as a substrate and measures the production of labeled adenosine 5'-phosphosulfate and 3'-phosphoadenosine 5'-phosphosulfate by low-voltage, hanging paper strip electrophoresis. The method is applicable to crude bacterial or mammalian extracts and accurately measures picomole amounts of product(s). Na2(75SeO4 can also be employed as a substrate, if the unstable radioactive product, adenosine 5'-phosphoselenate, is converted to elemental 75Se degrees by inclusion of reduced glutathione in the reaction mixture. The same paper strip electrophoretic technique can then be used to separate 75Se degrees from the radiolabeled substrate. The method also has utility for measuring any direct reduction by crude microbial extracts of radioactive selenate to selenite, independent of ATP sulfurylase.

Regional distribution of the phenylalanine-sensitive ATP-sulphurylase in brain.

The regional distribution of the phenylalanine-sensitive ATP-sulphurylase in fetal calf brain coincides with demyelinated lesions observed in the central nervous systems of untreated PKU patients. This would be expected if this species of ATP-sulphurylase played a role in the pathogenesis of brain dysfunction in the untreated or poorly controlled phenylketonuria patient.

The assay of ATP-sulfurylase.

An assay method for ATP-sulfurylase is described, based on the incorporation of 35SO4(2-) into adenosine-5'-phosphosulfate (and 3-phosphoadenosine-5'-phosphosulfate) and the separation of these compounds by HPLC. Since the enzyme is easily inactivated at temperatures above 20 degrees C during the assay, the reaction time should not exceed 10 min.

Comparative stability and catalytic and chemical properties of the sulfate-activating enzymes from Penicillium chrysogenum (mesophile) and Penicillium duponti (thermophile).

ATP sulfurylases from Penicillium chrysogenum (a mesophile) and from Penicillium duponti (a thermophile) had a native molecular weight of about 440,000 and a subunit molecular weight of about 69,000. (The P. duponti subunit appeared to be a little smaller than the P. chrysogenum subunit.) The P. duponti enzyme was about 100 times more heat stable than the P. chrysogenum enzyme; k inact (the first-order rate constant for inactivation) at 65 degrees C = 3.3 X 10(-4) s-1 for P. duponti and 3.0 X 10(-2) s-1 for P. chrysogenum. The P. duponti enzyme was also more stable to low pH and urea at 30 degrees C. Rabbit serum antibodies to each enzyme showed heterologous cross-reaction. Amino acid analyses disclosed no major compositional differences between the two enzymes. The analogous Km and Ki values of the forward and reverse reactions were also essentially identical at 30 degrees C. At 30 degrees C, the physiologically important adenosine 5'-phosphosulfate (APS) synthesis activity of the P. duponti enzyme was 4 U mg of protein-1, which is about half that of the P. chrysogenum enzyme. The molybdolysis and ATP synthesis activities of the P. duponti enzyme at 30 degrees C were similar to those of the P. chrysogenum enzyme. At 50 degrees C, the APS synthesis activity of the P. duponti enzyme was 12 to 19 U mg of protein-1, which was higher than that of the P. chrysogenum enzyme at 30 degrees C (8 +/- 1 U mg of protein-1). Treatment of the P. chrysogenum enzyme with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) at 30 degrees C under nondenaturing conditions modified one free sulfhydryl group per subunit. Vmax was not significantly altered, but the catalytic activity at low magnesium-ATP or SO4(2-) (or MoO4(2-)) was markedly reduced. Chemical modification with tetranitromethane had the same results on the kinetics. The native P. duponti enzyme was relatively unreactive toward DTNB or tetranitromethane at 30 degrees C and pH 8.0 or pH 9.0, but at 50 degrees C and pH 8.0, DTNB rapidly modified one SH group per subunit. APS kinase (the second sulfate-activating enzyme) of P. chrysogenum dissociated into inactive subunits at 42 degrees C. The P. duponti enzyme remained intact and active at 42 degrees C.

Kinetic and chemical properties of ATP sulphurylase from Penicillin chrysogenum.

Adenosine triphosphate sulphurylase (ATP: sulfate adenylyltransferase, EC 2.7.7.4.) has been purified from the filamentous fungus. Penicillium chrysogenum, and characterized physically, kinetically, and chemically. The P. Chrysogenum enzyme is an octomer (mol. wt. 440 000) composed of eight identical subunits (mol. wt. 55 000). Some physical constants are S20,w = 13.0 X 10(-13)s, D20,w = 2.94 X 10(-7) cm2 X s-1, v = 0.733 cm3 X g-1, A1%1cm = 8.71 at 278 nm. The enzyme catalyses (a) the synthesis of adenosine 5'-phosphosulphate (APS) and MgPPi from MgATP and SO2-4, (b) the hydrolysis of MgATP to AMP and MgPPi in the absence of SO2-4, (c) Mg32PPi-MgATP exchange in the absence of SO2-4, (d) molybdolysis of MgATP to AMP and MgPPi, (e) synthesis of MgATP and SO2-4 from APS and MgPPi, and (f) Mg32PPi-MgATP exchange in the presence of SO2-4. The Vmax values of reactions (a)-(c) are about 0.10-0.35 mumole X min-1 X mg enzyme-1. The Vmax values of reactions (d)-(f) are about 12-19 mumole X min-1 X mg enzyme-1. The catalytic activity of the enzyme in the direction of APS synthesis is rather low (0.13 unit X mg protein-1, corresponding to an active site turnover number of 7.15 min-1). However, the ATP sulphurylase content of mycelium growing on excess SO2-4 is 0.22 unit X g dry wt.-1, which is sufficient to account for the maximum in vivo rate of SO2-4 assimilation. The normal catalytic reaction is Ordered Bi Bi with A = MgATP, B = SO2-4, P = MgPPi, and Q = APS. Several lines of kinetic evidence suggest that the E.MgATP and E.APS complexes isomerize (to E approximately AMP.MgPPi and E approximately AMP.SO4, respectively) before the second substrate binds. Chemical modification studies have disclosed the presence of essential arginine, histidine, carboxyl, and tryosine residues. The latter is rather acidic (pKa = 7 or less). Nitration of the tyrosine increases the Km for MgATP without significantly affecting Kia for MgATP or Vmaxf. This result, and the fact that MgATP plus nitrate protects the enzyme against inactivation by tetranitromethane while MgATP alone does not, suggests that the essential tyrosine plays a role in nucleotide isomerization (perhaps as an adenylyl acceptor).