Crystal structure of phosphoadenylyl sulphate (PAPS) reductase: a new family of adenine nucleotide α hydrolases

Savage, H.; Montoya, Guillermo; Svensson, Cecilia; Schwenn, Jens D.; Sinning, Irmgard

doi:10.1016/s0969-2126(97)00244-x

Cited by 57 publications

(67 citation statements)

References 43 publications

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“…Previous results and independent verification by us shows that oxidized PAPR has a very low affinity for PAPS (SI Appendix, Fig. S7) (22). As expected, the presence of PAPS did not affect Trx binding to oxidized PAPR.…”

Section: Conformational Restriction Of Oxidized Papr Is Essential Forsupporting

confidence: 83%

“…3). A comparison of the crystal structures of apo-PAPR [Protein Data Bank (PDB) ID 1SUR], the noncovalent PAPR•PAP complex (PDB ID 2OQ2) and the covalent PAPR-Trx complex (PDB ID 2O8V), reveals significant differences in the active site architecture (17,22,23). For example, the otherwise disordered C-terminal tail residues (∼30 amino acids) including the catalytic cysteine (C239) fold over the substrate-binding pocket upon ligand binding (SI Appendix, Fig.…”

Section: (8)mentioning

confidence: 99%

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A universal entropy-driven mechanism for thioredoxin–target recognition

Palde

Carroll

2015

Proc. Natl. Acad. Sci. U.S.A.

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Cysteine residues in cytosolic proteins are maintained in their reduced state, but can undergo oxidation owing to posttranslational modification during redox signaling or under conditions of oxidative stress. In large part, the reduction of oxidized protein cysteines is mediated by a small 12-kDa thiol oxidoreductase, thioredoxin (Trx). Trx provides reducing equivalents for central metabolic enzymes and is implicated in redox regulation of a wide number of target proteins, including transcription factors. Despite its importance in cellular redox homeostasis, the precise mechanism by which Trx recognizes target proteins, especially in the absence of any apparent signature binding sequence or motif, remains unknown. Knowledge of the forces associated with the molecular recognition that governs Trx-protein interactions is fundamental to our understanding of target specificity. To gain insight into Trx-target recognition, we have thermodynamically characterized the noncovalent interactions between Trx and target proteins before S-S reduction using isothermal titration calorimetry (ITC). Our findings indicate that Trx recognizes the oxidized form of its target proteins with exquisite selectivity, compared with their reduced counterparts. Furthermore, we show that recognition is dependent on the conformational restriction inherent to oxidized targets. Significantly, the thermodynamic signatures for multiple Trx targets reveal favorable entropic contributions as the major recognition force dictating these proteinprotein interactions. Taken together, our data afford significant new insight into the molecular forces responsible for Trx-target recognition and should aid the design of new strategies for thiol oxidoreductase inhibition.thioredoxin | redox regulation | protein-protein interactions | entropy | oxidative stress

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Section: Conformational Restriction Of Oxidized Papr Is Essential Forsupporting

confidence: 83%

Section: (8)mentioning

confidence: 99%

A universal entropy-driven mechanism for thioredoxin–target recognition

Palde

Carroll

2015

Proc. Natl. Acad. Sci. U.S.A.

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“…The NTD of A. aeolicus TilS has the amino acid sequence 32 SGGVDS 37 . This sequence is identical to the fingerprint sequence, SGGXDss (where X is any hydrophobic amino acid and lowercase letters are highly conserved amino acids), of the ATP-binding P-loop in the N-type ATP-PPase subfamily, such as GMP synthetase, NAD synthetase, asparagine synthetase, and argininosuccinate synthetase, which commonly adenylates a substrate and then attacks the adenylated substrate with a nucleophilic nitrogen from a second substrate (16)(17)(18)(19). Furthermore, the catalytic domain of the ATP-PPase subfamily has been shown to adopt a dinucleotide-binding fold, which indicates that the TilS NTD was derived from the N-type ATP-PPase.…”

Section: Resultsmentioning

confidence: 95%

Structural basis for lysidine formation by ATP pyrophosphatase accompanied by a lysine-specific loop and a tRNA-recognition domain

Nakanishi

Fukai

Ikeuchi

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Lysidine, a lysine-combined modified cytidine, is exclusively located at the anticodon wobble position (position 34) of eubacterial tRNA Ile 2 and not only converts the codon specificity from AUG to AUA, but also converts the aminoacylation specificity from recognition by methionyl-tRNA synthetase to that by isoleucyl-tRNA synthetase (IleRS). Here, we report the crystal structure of lysidine synthetase (TilS) from Aquifex aeolicus at 2.42-Å resolution. TilS forms a homodimer, and each subunit consists of the N-terminal dinucleotide-binding fold domain (NTD), with a characteristic central hole, and the C-terminal globular domain (CTD) connected by a long ␣-helical linker. The NTD shares striking structural similarity with the ATP-pyrophosphatase domain of GMP synthetase, which reminds us of the two-step reaction by TilS: adenylation of C34 and lysine attack on the C2 carbon. Conserved amino acid residues are clustered around the NTD central hole. Kinetic analyses of the conserved residues' mutants indicated that C34 of tRNA Ile 2 is adenylated by an ATP lying across the NTD central hole and that a lysine, which is activated at a loop appended to the NTD, nucleophilically attacks the C2 carbon from the rear. Escherichia coli TilS (called MesJ) has an additional CTD, which may recognize the tRNA Ile 2 acceptor stem. In contrast, a mutational study revealed that A. aeolicus TilS does not recognize the tRNA acceptor stem but recognizes the C29⅐G41 base pair in the anticodon stem. Thus, the two TilS enzymes discriminate tRNA Ile 2 from tRNA Met by strategies similar to that used by IleRS, but in distinct manners.crystal structure ͉ isoleucyl-tRNA synthetase ͉ mutagenesis ͉ RNA modification

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“…Accordingly, APR and PAPR have nearly identical three-dimensional structures (1.6 Å root mean square deviation of backbone atoms) and share ϳ20% sequence identity, including the active site motif, ECG, and the sulfonucleotide binding pocket (5,11,12). However, a key difference between the two enzymes is that APR contains two conserved cysteine motifs: CXXC and CC.…”

Section: Mycobacterium Tuberculosis Adenosine 5-phosphosulfate Reductmentioning

confidence: 99%

“…Most proteins containing [4Fe-4S] clusters are redox-active (18 -21); however, the [4Fe-4S] 2ϩ cluster in APR does not undergo redox changes during the catalytic cycle (1). A purely structural role also appears unlikely in light of biophysical data obtained on the apo form of APR (6, 13, 16) and the fact that APR and PAPR share a common protein fold (5,11,12). Unfortunately, progress in this area has been hampered by the failure to generate a paramagnetic state of the [4Fe-4S] cluster that can be studied by EPR spectroscopy and related methods (16,17,22).…”

mentioning

confidence: 99%

Spectroscopic Studies on the [4Fe-4S] Cluster in Adenosine 5′-Phosphosulfate Reductase from Mycobacterium tuberculosis

Bhave¹,

Hong

Lee³

et al. 2011

Journal of Biological Chemistry

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Mycobacterium tuberculosis adenosine 5-phosphosulfate reductase (MtAPR) is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. The enzyme harbors a [4Fe-4S]2؉ cluster that is coordinated by four cysteinyl ligands, two of which are adjacent in the amino acid sequence. The ironsulfur cluster is essential for catalysis; however, the precise role of the [4Fe-4S] cluster in APR remains unknown. Progress in this area has been hampered by the failure to generate a paramagnetic state of the [4Fe-4S] cluster that can be studied by electron paramagnetic resonance spectroscopy. Herein, we overcome this limitation and report the EPR spectra of MtAPR in the [4Fe-4S] ؉ state. The EPR signal is rhombic and consists of two overlapping S ‫؍‬ 1 ⁄ 2 species. Substrate binding to MtAPR led to a marked increase in the intensity and resolution of the EPR signal and to minor shifts in principle g values that were not observed among a panel of substrate analogs, including adenosine 5-diphosphate. Using site-directed mutagenesis, in conjunction with kinetic and EPR studies, we have also identified an essential role for the active site residue Lys-144, whose side chain interacts with both the iron-sulfur cluster and the sulfate group of adenosine 5-phosphosulfate. The implications of these findings are discussed with respect to the role of the iron-sulfur cluster in the catalytic mechanism of APR.In bacteria and plants, activation of inorganic sulfur is required for de novo biosynthesis of cysteine. To this end, the metabolic assimilation of sulfate from the environment proceeds via adenosine 5Ј-phosphosulfate (APS) 2 or 3Ј-phosphoadenosine-5Ј-phosphosulfate (PAPS) (1). These intermediates are produced by the action of ATP-sulfurylase (EC 2.7.7.4), which condenses sulfate and ATP to form APS (2, 3), and by APS kinase (EC 2.7.1.25), which produces PAPS from ATP and APS (4).APS and PAPS are reduced by enzymes in the reductive branch of the sulfate assimilation pathway, producing sulfite and AMP or adenosine 3Ј,5Ј-diphosphate (Scheme 1). These enzymes can be subdivided into two groups according to their substrate preference: the APS reductases (APR) and the PAPS reductases (PAPR) (EC 1.8.99.4). Functional and structural studies have been used to investigate the chemical reaction mechanism of APR and PAPR enzymes (1,(5)(6)(7)(8). The mechanism involves nucleophilic attack by the active site cysteine on the sulfur atom of APS or PAPS to form an enzyme S-sulfocysteine intermediate, which is cleaved by thiol-disulfide exchange with thioredoxin or glutaredoxin (Fig. 1). The sulfite product is then reduced to sulfide by sulfite reductase (EC 1.8.7.1) and utilized to synthesize cysteine and other essential sulfur-containing biomolecules (9). In the human pathogen Mycobacterium tuberculosis, APR is a validated target against the latent phase of infection (10).Only a 3Ј-phosphate group distinguishes PAPS from APS. Accordingly, APR and PAPR have nearly identical three...

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Crystal structure of phosphoadenylyl sulphate (PAPS) reductase: a new family of adenine nucleotide α hydrolases

Cited by 57 publications

References 43 publications

A universal entropy-driven mechanism for thioredoxin–target recognition

A universal entropy-driven mechanism for thioredoxin–target recognition

Structural basis for lysidine formation by ATP pyrophosphatase accompanied by a lysine-specific loop and a tRNA-recognition domain

Spectroscopic Studies on the [4Fe-4S] Cluster in Adenosine 5′-Phosphosulfate Reductase from Mycobacterium tuberculosis

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