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Homology modeling of dissimilatory APS reductases (AprBA) of sulfur-oxidizing and sulfate-reducing prokaryotes.

Meyer B, Kuever J - PLoS ONE (2008)

Bottom Line: These structural alterations correlated with the protein phylogeny (three major phylogenetic lineages: (1) SRP including LGT-affected Archaeoglobi and SOB of Apr lineage II, (2) crenarchaeal SRP Caldivirga and Pyrobaculum, and (3) SOB of the distinct Apr lineage I) and the presence of potential APS reductase-interacting redox complexes.The almost identical protein matrices surrounding both [4Fe-4S] clusters, the FAD cofactor, the active site channel and center within the AprB/A models of SRP and SOB point to a highly similar catalytic process of APS reduction/sulfite oxidation independent of the metabolism type the APS reductase is involved in and the species it has been originated from.Based on the comparative models, there are no significant structural differences between dissimilatory APS reductases from SRP and SOB; this might be indicative for a similar catalytic process of APS reduction/sulfite oxidation.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Marine Microbiology, Bremen, Germany.

ABSTRACT

Background: The dissimilatory adenosine-5'-phosphosulfate (APS) reductase (cofactors flavin adenine dinucleotide, FAD, and two [4Fe-4S] centers) catalyzes the transformation of APS to sulfite and AMP in sulfate-reducing prokaryotes (SRP); in sulfur-oxidizing bacteria (SOB) it has been suggested to operate in the reverse direction. Recently, the three-dimensional structure of the Archaeoglobus fulgidus enzyme has been determined in different catalytically relevant states providing insights into its reaction cycle.

Methodology/principal findings: Full-length AprBA sequences from 20 phylogenetically distinct SRP and SOB species were used for homology modeling. In general, the average accuracy of the calculated models was sufficiently good to allow a structural and functional comparison between the beta- and alpha-subunit structures (78.8-99.3% and 89.5-96.8% of the AprB and AprA main chain atoms, respectively, had root mean square deviations below 1 A with respect to the template structures). Besides their overall conformity, the SRP- and SOB-derived models revealed the existence of individual adaptations at the electron-transferring AprB protein surface presumably resulting from docking to different electron donor/acceptor proteins. These structural alterations correlated with the protein phylogeny (three major phylogenetic lineages: (1) SRP including LGT-affected Archaeoglobi and SOB of Apr lineage II, (2) crenarchaeal SRP Caldivirga and Pyrobaculum, and (3) SOB of the distinct Apr lineage I) and the presence of potential APS reductase-interacting redox complexes. The almost identical protein matrices surrounding both [4Fe-4S] clusters, the FAD cofactor, the active site channel and center within the AprB/A models of SRP and SOB point to a highly similar catalytic process of APS reduction/sulfite oxidation independent of the metabolism type the APS reductase is involved in and the species it has been originated from.

Conclusions: Based on the comparative models, there are no significant structural differences between dissimilatory APS reductases from SRP and SOB; this might be indicative for a similar catalytic process of APS reduction/sulfite oxidation.

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AprA active center from the protein of A. fulgidus (A, B) and the homology modeling-based model of Pyrobaculum calidifontis (C, D) (residues in a distance of less than 6.5 Å to the N5 atom of FAD cofactor are shown).FAD cofactor and substrate APS (in two conformations) are shown as ball-and-stick representations. Residues involved in the isoalloxazine binding (e.g. A. fulgidus: Leu-A70, Asn-A74, Trp-A234) are highlighted by green color (missing Asn-A63 in the Pyrobaculum calidifontis AprA model is marked by an arrow), the invariant, positively charged residues His-A398 and Arg-A265 of the active site are blue colored (other AA are colored in grey). Distances are given in Å (B, D). (E) The position of the electron-transferring [4Fe-4S] cluster I and Trp-B48 (highlighted by violet color) of AprB to the FAD cofactor in the AprA protein of A. fulgidus is shown (ribbon structure is colored in grey).
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pone-0001514-g009: AprA active center from the protein of A. fulgidus (A, B) and the homology modeling-based model of Pyrobaculum calidifontis (C, D) (residues in a distance of less than 6.5 Å to the N5 atom of FAD cofactor are shown).FAD cofactor and substrate APS (in two conformations) are shown as ball-and-stick representations. Residues involved in the isoalloxazine binding (e.g. A. fulgidus: Leu-A70, Asn-A74, Trp-A234) are highlighted by green color (missing Asn-A63 in the Pyrobaculum calidifontis AprA model is marked by an arrow), the invariant, positively charged residues His-A398 and Arg-A265 of the active site are blue colored (other AA are colored in grey). Distances are given in Å (B, D). (E) The position of the electron-transferring [4Fe-4S] cluster I and Trp-B48 (highlighted by violet color) of AprB to the FAD cofactor in the AprA protein of A. fulgidus is shown (ribbon structure is colored in grey).

Mentions: The reductive reaction mechanism of the dissimilatory APS reductase proceeds via a nucleophilic attack of the N5 atom (isoalloxazine moiety) of the reduced FAD on the sulfur of APS [18], [23], [24]. According to the AprA comparative models, 35 to 36 mostly conserved amino acids are located at a distance of less than 4.1 Å to the FAD cofactor (comprising residues of sec. str. elm. 1 to 2, 3 to 4, 11, 14 to 15, 16 to 18, 31 to 32, and 35 to 36 including the interjacent loop regions, see Fig. 8; for details see also supplementary data material Figure S3, panels G/H, and supplementary data material Table S4); thus, the cofactor-surrounding protein matrices are highly conserved among the APS reductases of SOB and SRP. As revealed by the A. fulgidus 3D protein structure, a pronounced feature of the FADH− state in the enzyme is the substantial bend of the isoalloxazine ring along the N5-N10 axis by an angle of 25° (Fig. 9 and 10). In general, the protein matrix could have a considerable influence on the bending angle of the latter and thereby affect the redox potential of FAD; a flat conformation of FAD (0–10°) favors the oxidized state and a bent “butterfly” conformation (15–30°) favors the reduced state. In the A. fulgidus APS reductase, the side chains of Asn-A74 and Trp-A234 that are located at the re-face of FAD enforce a shift of the dimethylbenzene and pyrimidine rings toward the si-face of the FAD, whereas the pyrazine ring is held in position by Leu-A70 which protrudes toward the si-face of FAD (Fig. 9 panels A and B). The induced stabilization of the reduced form of FAD agrees with the experimentally determined higher reduction potentials of ∼−45 mV in APS reductases of several SRP compared to ∼−220 mV of free FAD [18], [23], [24]. Notably, the aforementioned residues are strictly conserved and strongly fixed at their positions in all AprA models of the sulfate reducers (except Pyrobaculum calidifontis, see Fig. 9 panels C and D) and even the sulfur-oxidizers (see supplementary data material Figure S4 for details) which might indicate that this cofactor is also present in a bent conformation in the SOB-type APS reductases although the coplanar arrangement of the three aromatic rings would be the energetical favorable conformation in the oxidized state. Indeed, the isoalloxazine ring system of A. fulgidus APS reductase revealed an identical bending angle in the oxidized as in the reduced FAD state [18], [23], [24] i.e. the butterfly conformation is maintained in the A. fulgidus protein independent of the redox potential of the FAD cofactor and direction of catalytic reaction (Fig. 10). This is also demonstrated by the AprA models of this study that encompassed reductive and oxidative type APS reductases from distinct SRP and SOB. In contrast, other enzymes e.g. thioredoxin reductase [48], alternate the FAD conformation with respect to their oxidized and reduced states. The AprA model of Pyrobaculum calidifontis lacked the structurally essential Asn-A63 at the re-face of FAD (see Fig. 9 panels C and D). However, the strained conformation of the isoalloxazine moiety was suggested to be important for efficient electron flow between the redox centers by facilitating the reduction of the oxidized FAD via the [4Fe-4S] clusters. In agreement with the AprB model-derived results, this missing structural feature might be another indication for the non-functionality of the APS reductase present in the investigated Pyrobaculum species.


Homology modeling of dissimilatory APS reductases (AprBA) of sulfur-oxidizing and sulfate-reducing prokaryotes.

Meyer B, Kuever J - PLoS ONE (2008)

AprA active center from the protein of A. fulgidus (A, B) and the homology modeling-based model of Pyrobaculum calidifontis (C, D) (residues in a distance of less than 6.5 Å to the N5 atom of FAD cofactor are shown).FAD cofactor and substrate APS (in two conformations) are shown as ball-and-stick representations. Residues involved in the isoalloxazine binding (e.g. A. fulgidus: Leu-A70, Asn-A74, Trp-A234) are highlighted by green color (missing Asn-A63 in the Pyrobaculum calidifontis AprA model is marked by an arrow), the invariant, positively charged residues His-A398 and Arg-A265 of the active site are blue colored (other AA are colored in grey). Distances are given in Å (B, D). (E) The position of the electron-transferring [4Fe-4S] cluster I and Trp-B48 (highlighted by violet color) of AprB to the FAD cofactor in the AprA protein of A. fulgidus is shown (ribbon structure is colored in grey).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211403&req=5

pone-0001514-g009: AprA active center from the protein of A. fulgidus (A, B) and the homology modeling-based model of Pyrobaculum calidifontis (C, D) (residues in a distance of less than 6.5 Å to the N5 atom of FAD cofactor are shown).FAD cofactor and substrate APS (in two conformations) are shown as ball-and-stick representations. Residues involved in the isoalloxazine binding (e.g. A. fulgidus: Leu-A70, Asn-A74, Trp-A234) are highlighted by green color (missing Asn-A63 in the Pyrobaculum calidifontis AprA model is marked by an arrow), the invariant, positively charged residues His-A398 and Arg-A265 of the active site are blue colored (other AA are colored in grey). Distances are given in Å (B, D). (E) The position of the electron-transferring [4Fe-4S] cluster I and Trp-B48 (highlighted by violet color) of AprB to the FAD cofactor in the AprA protein of A. fulgidus is shown (ribbon structure is colored in grey).
Mentions: The reductive reaction mechanism of the dissimilatory APS reductase proceeds via a nucleophilic attack of the N5 atom (isoalloxazine moiety) of the reduced FAD on the sulfur of APS [18], [23], [24]. According to the AprA comparative models, 35 to 36 mostly conserved amino acids are located at a distance of less than 4.1 Å to the FAD cofactor (comprising residues of sec. str. elm. 1 to 2, 3 to 4, 11, 14 to 15, 16 to 18, 31 to 32, and 35 to 36 including the interjacent loop regions, see Fig. 8; for details see also supplementary data material Figure S3, panels G/H, and supplementary data material Table S4); thus, the cofactor-surrounding protein matrices are highly conserved among the APS reductases of SOB and SRP. As revealed by the A. fulgidus 3D protein structure, a pronounced feature of the FADH− state in the enzyme is the substantial bend of the isoalloxazine ring along the N5-N10 axis by an angle of 25° (Fig. 9 and 10). In general, the protein matrix could have a considerable influence on the bending angle of the latter and thereby affect the redox potential of FAD; a flat conformation of FAD (0–10°) favors the oxidized state and a bent “butterfly” conformation (15–30°) favors the reduced state. In the A. fulgidus APS reductase, the side chains of Asn-A74 and Trp-A234 that are located at the re-face of FAD enforce a shift of the dimethylbenzene and pyrimidine rings toward the si-face of the FAD, whereas the pyrazine ring is held in position by Leu-A70 which protrudes toward the si-face of FAD (Fig. 9 panels A and B). The induced stabilization of the reduced form of FAD agrees with the experimentally determined higher reduction potentials of ∼−45 mV in APS reductases of several SRP compared to ∼−220 mV of free FAD [18], [23], [24]. Notably, the aforementioned residues are strictly conserved and strongly fixed at their positions in all AprA models of the sulfate reducers (except Pyrobaculum calidifontis, see Fig. 9 panels C and D) and even the sulfur-oxidizers (see supplementary data material Figure S4 for details) which might indicate that this cofactor is also present in a bent conformation in the SOB-type APS reductases although the coplanar arrangement of the three aromatic rings would be the energetical favorable conformation in the oxidized state. Indeed, the isoalloxazine ring system of A. fulgidus APS reductase revealed an identical bending angle in the oxidized as in the reduced FAD state [18], [23], [24] i.e. the butterfly conformation is maintained in the A. fulgidus protein independent of the redox potential of the FAD cofactor and direction of catalytic reaction (Fig. 10). This is also demonstrated by the AprA models of this study that encompassed reductive and oxidative type APS reductases from distinct SRP and SOB. In contrast, other enzymes e.g. thioredoxin reductase [48], alternate the FAD conformation with respect to their oxidized and reduced states. The AprA model of Pyrobaculum calidifontis lacked the structurally essential Asn-A63 at the re-face of FAD (see Fig. 9 panels C and D). However, the strained conformation of the isoalloxazine moiety was suggested to be important for efficient electron flow between the redox centers by facilitating the reduction of the oxidized FAD via the [4Fe-4S] clusters. In agreement with the AprB model-derived results, this missing structural feature might be another indication for the non-functionality of the APS reductase present in the investigated Pyrobaculum species.

Bottom Line: These structural alterations correlated with the protein phylogeny (three major phylogenetic lineages: (1) SRP including LGT-affected Archaeoglobi and SOB of Apr lineage II, (2) crenarchaeal SRP Caldivirga and Pyrobaculum, and (3) SOB of the distinct Apr lineage I) and the presence of potential APS reductase-interacting redox complexes.The almost identical protein matrices surrounding both [4Fe-4S] clusters, the FAD cofactor, the active site channel and center within the AprB/A models of SRP and SOB point to a highly similar catalytic process of APS reduction/sulfite oxidation independent of the metabolism type the APS reductase is involved in and the species it has been originated from.Based on the comparative models, there are no significant structural differences between dissimilatory APS reductases from SRP and SOB; this might be indicative for a similar catalytic process of APS reduction/sulfite oxidation.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Marine Microbiology, Bremen, Germany.

ABSTRACT

Background: The dissimilatory adenosine-5'-phosphosulfate (APS) reductase (cofactors flavin adenine dinucleotide, FAD, and two [4Fe-4S] centers) catalyzes the transformation of APS to sulfite and AMP in sulfate-reducing prokaryotes (SRP); in sulfur-oxidizing bacteria (SOB) it has been suggested to operate in the reverse direction. Recently, the three-dimensional structure of the Archaeoglobus fulgidus enzyme has been determined in different catalytically relevant states providing insights into its reaction cycle.

Methodology/principal findings: Full-length AprBA sequences from 20 phylogenetically distinct SRP and SOB species were used for homology modeling. In general, the average accuracy of the calculated models was sufficiently good to allow a structural and functional comparison between the beta- and alpha-subunit structures (78.8-99.3% and 89.5-96.8% of the AprB and AprA main chain atoms, respectively, had root mean square deviations below 1 A with respect to the template structures). Besides their overall conformity, the SRP- and SOB-derived models revealed the existence of individual adaptations at the electron-transferring AprB protein surface presumably resulting from docking to different electron donor/acceptor proteins. These structural alterations correlated with the protein phylogeny (three major phylogenetic lineages: (1) SRP including LGT-affected Archaeoglobi and SOB of Apr lineage II, (2) crenarchaeal SRP Caldivirga and Pyrobaculum, and (3) SOB of the distinct Apr lineage I) and the presence of potential APS reductase-interacting redox complexes. The almost identical protein matrices surrounding both [4Fe-4S] clusters, the FAD cofactor, the active site channel and center within the AprB/A models of SRP and SOB point to a highly similar catalytic process of APS reduction/sulfite oxidation independent of the metabolism type the APS reductase is involved in and the species it has been originated from.

Conclusions: Based on the comparative models, there are no significant structural differences between dissimilatory APS reductases from SRP and SOB; this might be indicative for a similar catalytic process of APS reduction/sulfite oxidation.

Show MeSH
Related in: MedlinePlus