Purification and characterization of ATP sulfurylase from the extremely thermophilic archaebacterial sulfate-reducer, Archaeoglobus fulgidus (original) (raw)
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FEMS Microbiology Letters, 1998
In the hyperthermophilic sulfate reducer Archaeoglobus fulgidus DSM 4304 , two open reading frames (sat and ORF2) are located upstream of the aprBA genes encoding adenosine-5P-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 sulfuroxidizing 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. z 1998 Published by Elsevier Science B.V.
Proceedings of the National Academy of Sciences, 2002
The iron-sulfur flavoenzyme adenylylsulfate (adenosine 5′-phosphosulfate, APS) reductase catalyzes reversibly the reduction of APS to sulfite and AMP. The structures of APS reductase from the hyperthermophilic Archaeoglobus fulgidus in the two-electron reduced state and with sulfite bound to FAD are reported at 1.6- and 2.5-Å resolution, respectively. The FAD-sulfite adduct was detected after soaking the crystals with APS. This finding and the architecture of the active site strongly suggest that catalysis involves a nucleophilic attack of the N5 atom of reduced FAD on the sulfur atom of APS. In view of the high degree of similarity between APS reductase and fumarate reductase especially with regard to the FAD-binding α-subunit, it is proposed that both subunits originate from a common ancestor resembling archaeal APS reductase. The two electrons required for APS reduction are transferred via two [4Fe-4S] clusters from the surface of the protein to FAD. The exceptionally large diffe...
Archives of Biochemistry and Biophysics, 1983
Homogeneous ATP sulfurylase from Penicillium chrysogenum has been reported to have an extremely low activity toward its physiological inorganic substrate, sulfate. This low activity is an artifact resulting from potent product inhibition by 5'-adenylylsulfate (APS) (Ki < 0.25 PM). Assays based on 35S incorporation from 35S042-into charcoal-adsorbable [%]APS are nonlinear with time, even in the presence of a large excess of inorganic pyrophosphatase. However, in the presence of excess APS kinase (along with excess pyrophosphatase), the ATP sulfurylase reaction is linear with time and the enzyme has a specific activity (V,,,) of 6 to '7 units mg protein-' corresponding to an active site turnover number of at least 400 min-'. Monovalent oxyanions such as N03-, C103-, C104-, and FS03-are competitive with sulfate (or molybdate) and essentially uncompetitive with respect to MgATP. However, thiosulfate (SS032-), a true sulfate analog and dead-end inhibitor of the enzyme (competitive with sulfate or molybdate), exhibited clear noncompetitive inhibition against MgATP. Furthermore, APS was competitive with both MgATP and molybdate in the molybdolysis assay. These results suggest (a) that the mechanism of the normal forward reaction may be random rather than ordered and (b) that the monovalent oxyanions have a much greater affinity for the E. MgATP complex than for free E. In this respect, FS03-, C104-, etc., are not true sulfate analogs although they might mimic an enzyme-bound species formed when MgATP is at the active site. The nonlinear ATP sulfurylase reaction progress curves (with APS accumulating in the presence of excess pyrophosphatase OT PPi accumulating in the presence of excess APS kinase) were analyzed by means of "average velocity" plots based on an integrated rate equation. This new approach is useful for enzymes subject to potent product inhibition over a reaction time course in which the substrate concentrations do not change significantly. The analysis showed that ATP sulfurylase has an intrinsic specific activity of 6 to 7 units mg protein-'. Thus, the apparent stimulation of sulfurylase activity by APS kinase results from the continual removal of inhibitory APS rather than from an association of the two sulfate-activating enzymes to form a "3'-phospho-5'-adenylylsulfate synthetase" complex in which the sulfurylase has an increased catalytic activity. The progress curve analyses suggest that APS is competitive with both MgATP and sulfate, while MgPPi is a mixed-type inhibitor with respect to both substrates. The cumulative data point to a random sequence for the forward reaction with APS release being partially rate limiting.
Biological chemistry Hoppe-Seyler, 1988
A modified procedure for the purification of soluble ATPase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius is described. In addition to (a) 65 and ( ) 51 kDa polypeptides, further subunits 7* (20 kDa) and 5* (12 kDa) are demonstrated to be components of the enzyme, exhibiting a total molecular mass of 380 kDa. Molecular electron microscopic images of the native enzyme indicate a quaternary structure probably formed by the 7*, *complex as a central mass surrounded by a pseu-dohexagon of the peripherally arranged larger and subunits. As can be derived from both molecular mass and electron microscopy data, the archaebacterial Sulfolobus-ATPs.se emerges to exist as an : 3 3 -quaternary structure with respect to the larger subunits. This is normally found in typical Fj-ATPases of eubacterial and eukaryotic organisms. Therefore it is postulated that F r and FoFj-ATPases, respectively, can occur ubiquitously in all urkingdoms of organisms as functional units of energy-transducing membranes. Archaebakterien-ATPase: Untersuchungen über die Untereinheiten-Zusammensetzung und Quartärstruktur der Fj-analogen ATPase aus Sulfolobus acidocaldarius Zusammenfassung: Ein modifiziertes Verfahren zur Reinigung der löslichen ATPase aus dem thermoacidophilen Archaebakterium Sulfolobus acidocaldarius wird beschrieben. Es wird gezeigt, daß neben den früher gefundenen* 65 kDa (a-) und 51 kDa (ß-) Polypeptiden die weiteren Untereinheiten 7* (20 kDa) und * (12 kDa) zum Enzym gehören, das insgesamt eine molekulare Masse von 380 kDa aufweist. Elektronenmikroskopische Aufnahmen des nativen Enzymmoleküls deuten eine pseudohexagonale Quartärstruktur an, die wahrscheinlich aus dem von den peripher angeordneten großen a-und ß-Unter-einheiten umgebenen zentralen 7*,6*-Komplex gebildet wird. Wie sich aus den kombinierten Elektronenmikroskopie-und Molekularmassen-Daten ableiten läßt, muß für die Sulfolobus-ATPase eine a 3 j3 3 -Stöchiometrie der großen Untereinheiten angenommen werden. Diese wird normalerweise bei typischen F r ATPasen eubakterieller und eukaryontischer Organismen gefunden. Es wird daher postuliert, daß F r , bzw. F 0 F r ATPasen ubiquitär in allen Organismenreichen als Funktionseinheiten energieumwandelnder Membransysteme vorkommen kön-
Gene, 1996
The gene from the thermophilic archaeon Sulfolobus solfataricus (Ss), encoding the S-adenosylhomocysteine hydrolase (AdoHcyHD), has been cloned. Two degenerate oligodeoxyribonucleotide (oligo) probes, synthesized on the basis of amino acid (aa) sequence of cyanogen bromide-peptide fragments of the purified protein, were used to screen a genomic library of Ss cloned into the pGEM7Zf(+) vector. The AdoHcyHD gene (adohcyhd) comprises 1254 nucleotides (nt) and encodes a polypeptide of 417 aa with a deduced molecular mass of 46 kDa, in good agreement with the value directly measured for the purified enzyme. The identity of more than 32% of the deduced aa sequence was confirmed by Edman degradation of peptides. Putative regulatory elements which are in good agreement with the archaeal promoter consensus sequences were identified in the flanking regions. Comparison of the aa sequences of AdoHcyHD from different sources shows a remarkable degree of conservation. Surprisingly, several aa residues, thought important in substrate binding and catalysis, show non-conserved replacements in Ss AdoHcyHD.