Structure of adenylylsulfate reductase from the hyperthermophilic Archaeoglobus fulgidus at 1.6-Å resolution (original) (raw)
Related papers
Biochemistry, 2006
The iron-sulfur flavoenzyme adenosine-5′-phosphosulfate (APS) reductase catalyzes a key reaction of the global sulfur cycle by reversibly transforming APS to sulfite and AMP. The structures of the dissimilatory enzyme from Archaeoglobus fulgidus in the reduced state (FAD red) and in the sulfite adduct state (FAD-sulfite-AMP) have been recently elucidated at 1.6 and 2.5 Å resolution, respectively. Here we present new structural features of the enzyme trapped in four different catalytically relevant states that provide us with a detailed picture of its reaction cycle. In the oxidized state (FAD ox), the isoalloxazine moiety of the FAD cofactor exhibits a similarly bent conformation as observed in the structure of the reduced enzyme. In the APS-bound state (FAD ox-APS), the substrate APS is embedded into a 17 Å long substrate channel in such a way that the isoalloxazine ring is pushed toward the channel bottom, thereby producing a compressed enzyme-substrate complex. A clamp formed by residues ArgA317 and LeuA278 to fix the adenine ring and the curved APS conformation appear to be key factors to hold APS in a strained conformation. This energy-rich state is relaxed during the attack of APS on the reduced FAD. A relaxed FAD-sulfite adduct is observed in the structure of the FAD-sulfite state. Finally, a FAD-sulfite-AMP1 state with AMP within van der Waals distance of the sulfite adduct could be characterized. This structure documents how adjacent negative charges are stabilized by the protein matrix which is crucial for forming APS from AMP and sulfite in the reverse reaction.
Microbiology, 1994
Adenylylsulphate (adenosine-5'-phosphosulphate, APS) reductase from the extremely thermophilic sulphate-reducing archaeon Archaeoglobus fulgidus is an iron-sulphur f lavoprotein containing one non-covalently bound f lavin group, eight non-haem iron and six labile sulphide atoms per molecule. Reevaluation of the enzyme structure revealed the presence of two different subunits with molecular masses of 80 and 18.5 kDa. The subunits are arranged in an a# subunit structure. We have cloned and sequenced a 2-7 kb segment of DNA containing the genes for the a and p subunits, which we designate aprA and aprB, respectively. The two genes are separated by 17 bp and localized in the order aprBA. While a putative promoter could not be identified in the vicinity of aprBA a probable termination signal was found just downstream of the translation stop codon of aprA. The codon usage for aprBA shows strong preferences for G and C in the third codon position. aprA encodes a 73.3 kDa polypeptide, which shows significant overall similarities with the flavoprotein subunits of the succinate dehydrogenases from Escherichia coli and Bacillus subtilis and the corresponding f lavoprotein of E. coli f umarate reductase. Part of the homologous peptide stretches could be assigned to domains that are involved in the binding of the substrate or of the FAD prosthetic group. aprB encodes a 17.1 kDa polypeptide representing an iron-sulphur protein, seven cysteine residues of which are arranged in two clusters typical of ligands of the iron-sulphur centres in { [ Fe, S, ] [ Fe, S, ] } 7-Fe ferredoxins.
Journal of Bacteriology, 2009
Adenylylsulfate reductase (adenosine 5′-phosphosulfate [APS] reductase [APSR]) plays a key role in catalyzing APS to sulfite in dissimilatory sulfate reduction. Here, we report the crystal structure of APSR from Desulfovibrio gigas at 3.1-Å resolution. Different from the α 2 β 2 -heterotetramer of the Archaeoglobus fulgidus , the overall structure of APSR from D. gigas comprises six αβ-heterodimers that form a hexameric structure. The flavin adenine dinucleotide is noncovalently attached to the α-subunit, and two [4Fe-4S] clusters are enveloped by cluster-binding motifs. The substrate-binding channel in D. gigas is wider than that in A. fulgidus because of shifts in the loop (amino acid 326 to 332) and the α-helix (amino acid 289 to 299) in the α-subunit. The positively charged residue Arg160 in the structure of D. gigas likely replaces the role of Arg83 in that of A. fulgidus for the recognition of substrates. The C-terminal segment of the β-subunit wraps around the α-subunit to f...
Journal of Bacteriology, 2009
catalyzing APS to sulfite in dissimilatory sulfate reduction. Here, we report the crystal structure of APSR from Desulfovibrio gigas at 3.1-Å resolution. Different from the ␣ 2  2 -heterotetramer of the Archaeoglobus fulgidus, the overall structure of APSR from D. gigas comprises six ␣-heterodimers that form a hexameric structure. The flavin adenine dinucleotide is noncovalently attached to the ␣-subunit, and two [4Fe-4S] clusters are enveloped by cluster-binding motifs. The substrate-binding channel in D. gigas is wider than that in A. fulgidus because of shifts in the loop (amino acid 326 to 332) and the ␣-helix (amino acid 289 to 299) in the ␣-subunit. The positively charged residue Arg160 in the structure of D. gigas likely replaces the role of Arg83 in that of A. fulgidus for the recognition of substrates. The C-terminal segment of the -subunit wraps around the ␣-subunit to form a functional unit, with the C-terminal loop inserted into the active-site channel of the ␣-subunit from another ␣-heterodimer. Electrostatic interactions between the substrate-binding residue Arg282 in the ␣-subunit and Asp159 in the C terminus of the -subunit affect the binding of the substrate. Alignment of APSR sequences from D. gigas and A. fulgidus shows the largest differences toward the C termini of the -subunits, and structural comparison reveals notable differences at the C termini, activity sites, and other regions. The disulfide comprising Cys156 to Cys162 stabilizes the C-terminal loop of the -subunit and is crucial for oligomerization. Dynamic light scattering and ultracentrifugation measurements reveal multiple forms of APSR upon the addition of AMP, indicating that AMP binding dissociates the inactive hexamer into functional dimers, presumably by switching the C terminus of the -subunit away from the active site. The crystal structure of APSR, together with its oligomerization properties, suggests that APSR from sulfatereducing bacteria might self-regulate its activity through the C terminus of the -subunit.
European Journal of Biochemistry, 1990
In order to utilize sulfate as the terminal electron acceptor, sulfate-reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (AdoPSO,) reductase plays one of the major roles, reducing AdoPS04 (the activated form of sulfate) to sulfite, with release of AMP. The enzyme has been purified to homogeneity from the anaerobic sulfate reducer Desulfovihrio gigas. The protein is composed of two non-identical subunits (70 kDa and 23 kDa) and is isolated in a multimeric form ( z 400 kDa). It is an iron-sulfur, flavincontaining protein, with one FAD moiety, eight iron atoms and a minimum molecular mass of 93 kDa.
The active centers of adenylylsulfate reductase from Desulfovibrio gigas
1990
In order to utilize sulfate as the terminal electron acceptor, sulfate-reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (AdoPSO,) reductase plays one of the major roles, reducing AdoPS04 (the activated form of sulfate) to sulfite, with release of AMP. The enzyme has been purified to homogeneity from the anaerobic sulfate reducer Desulfovihrio gigas. The protein is composed of two non-identical subunits (70 kDa and 23 kDa) and is isolated in a multimeric form (z 400 kDa). It is an iron-sulfur, flavincontaining protein, with one FAD moiety, eight iron atoms and a minimum molecular mass of 93 kDa. Low-temperature EPR studies were performed to characterize its redox centers. In the native state, the enzyme showed an almost isotropic signal centered at g = 2.02 and only detectable below 20 K. This signal represented a minor species (0.10-0.25 spins/mol) and showed line broadening in the enzyme isolated from 57Fe-grown cells. Addition of sulfite had a minor effect on the EPR spectrum, but caused a major decrease in the visible region of the optical spectrum (around 392 nm). Further addition of AMP induced only a minor change in the visible spectrum whereas major changes were seen in the EPR spectrum; the appearance of a rhombic signal at g values 2.096, 1.940 and 1.890 (reduced Fe-S center I) observable below 30 K and a concomitant decrease in intensity of the g = 2.02 signal were detected. Effects of chemical reductants (ascorbate, H,/hydrogenase-reduced methyl viologen and dithionite) were also studied. A short time reduction with dithionite (15 s) or reduction with methyl viologen gave rise to the full reduction of center I (with slightly modified g values at 2.079, 1.939 and 1.897), and the complete disappearance of the g = 2.02 signal. Further reduction with dithionite produces a very complex EPR spectrum of a spin-spin-coupled nature (observable below 20 K), indicating the presence of at least two iron-sulfur centers, (centers I and 11). Mossbauer studies on "Fe-enriched D. gigas Ado P S 0 4 reductase demonstrated unambiguously the presence of two 4Fe clusters. Center I1 has a redox potential < 400 mV and exhibits spectroscopic properties that are Biochim. Biophys. Acta 537, 255-269.