Characterization and Reconstitution of a 4Fe-4S Adenylyl Sulfate/Phosphoadenylyl Sulfate Reductase from Bacillus subtilis

Berndt, Carsten; Lillig, Christopher Horst; Wollenberg, Markus; Bill, Eckhard; Mansilla, María C.; Mendoza, Diego de; Seidler, Andreas; Schwenn, Jens D.

doi:10.1074/jbc.m309332200

Cited by 60 publications

(75 citation statements)

References 31 publications

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“…Sulfate is first transported into the cell via a sulfate permease, CysP, related to inorganic phosphate transporters (26). Sulfate is subsequently reduced to sulfide, probably in four steps involving the sequential action of ATP sulfurylase, adenosine 5Ј-phosphosulfate (APS) kinase, 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS) reductase, and sulfite reductase (3,27,55). An O-acetylserine thiol-lyase, the cysK gene product, further condenses sulfide and O-acetylserine (OAS) to form cysteine (55).…”

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confidence: 99%

Global Control of Cysteine Metabolism by CymR inBacillus subtilis

et al. 2006

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YrzC has previously been identified as a repressor controlling ytmI expression via its regulation of YtlI activator synthesis in Bacillus subtilis. We identified YrzC as a master regulator of sulfur metabolism. Gene expression profiles of B. subtilis ⌬yrzC mutant and wild-type strains grown in minimal medium with sulfate as the sole sulfur source were compared. In the mutant, increased expression was observed for 24 genes previously identified as repressed in the presence of sulfate. Since several genes involved in the pathways leading to cysteine formation were found, we propose to rename YrzC CymR, for "cysteine metabolism repressor." A CymR-dependent binding to the promoter region of the ytlI, ssuB, tcyP, yrrT, yxeK, cysK, or ydbM gene was demonstrated using gel shift experiments. A potential CymR target site, TAAWNCN 2 ANTWNAN 3 ATMGGAA TTW, was found in the promoter region of these genes. In a DNase footprint experiment, the protected region in the ytlI promoter region contained this consensus sequence. Partial deletion or introduction of point mutations in this sequence confirmed its involvement in ytlI, yrrT, and yxeK regulation. The addition of O-acetylserine in gel shift experiments prevented CymR-dependent binding to DNA for all of the targets characterized. Transcriptome analysis of a ⌬cymR mutant and the wild-type strain also brought out significant changes in the expression level of a large set of genes related to stress response or to transition toward anaerobiosis.

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Global Control of Cysteine Metabolism by CymR inBacillus subtilis

et al. 2006

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“…In Bacillus subtilis, sulfate is transported into the cell via a sulfate permease, CysP, related to inorganic phosphate transporters (24). Sulfate is subsequently reduced to sulfide probably in four steps involving ATP sulfurylase, adenosine 5Ј-phosphosulfate (APS) kinase, 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS) reductase, and sulfite reductase (3,25,45). An O-acetyl-L-serine thiol-lyase, the cysK gene product, condenses sulfide and Oacetylserine (OAS) to form cysteine (45).…”

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confidence: 99%

Regulation of the Bacillus subtilis ytmI Operon, Involved in Sulfur Metabolism

et al. 2005

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The YtlI regulator of Bacillus subtilis activates the transcription of the ytmI operon encoding an L-cystine ABC transporter, a riboflavin kinase, and proteins of unknown function. The expression of the ytlI gene and the ytmI operon was high with methionine and reduced with sulfate. Using deletions and site-directed mutagenesis, a cis-acting DNA sequence important for YtlI-dependent regulation was identified upstream from the ؊35 box of ytmI. Gel mobility shift assays confirmed that YtlI specifically interacted with this sequence. The replacement of the sulfur-regulated ytlI promoter by the xylA promoter led to constitutive expression of a ytmI-lacZ fusion in a ytlI mutant, suggesting that the repression of ytmI expression by sulfate was mainly at the level of YtlI synthesis. We further showed that the YrzC regulator negatively controlled ytlI expression while this repressor also acted on ytmI expression via YtlI. The cascade of regulation observed in B. subtilis is conserved in Listeria spp. Both a YtlI-like regulator and a ytmI-type operon are present in Listeria spp. Indeed, the Lmo2352 protein from Listeria monocytogenes was able to replace YtlI for the activation of ytmI expression and a lmo2352-lacZ fusion was repressed in the presence of sulfate via YrzC in B. subtilis. A common motif, AT(A/T)ATTCCTAT, was found in the promoter region of the ytlI and lmo2352 genes. Deletion of part of this motif or the introduction of point mutations in this sequence confirmed its involvement in ytlI regulation.All living organisms require sulfur for the synthesis of proteins and essential cofactors. Sulfur can be assimilated either from inorganic sources, such as sulfate and thiosulfate, or from organic sources, such as sulfate esters, sulfamates, sulfonates, cysteine, methionine, or their derivatives. In Bacillus subtilis, sulfate is transported into the cell via a sulfate permease, CysP, related to inorganic phosphate transporters (24). Sulfate is subsequently reduced to sulfide probably in four steps involving ATP sulfurylase, adenosine 5Ј-phosphosulfate (APS) kinase, 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS) reductase, and sulfite reductase (3,25,45). An O-acetyl-L-serine thiol-lyase, the cysK gene product, condenses sulfide and Oacetylserine (OAS) to form cysteine (45). Sulfonates, cysteine, and methionine can be used as sulfur sources by B. subtilis. Several alkanesulfonates are taken up by a sulfonate ABC (ATP-binding cassette) transporter and then converted to sulfite by a FMNH 2 -dependent monooxygenase (46). The transport of L-cystine, the oxidized form of cysteine, has recently been investigated. Three systems are present in B. subtilis: two ABC transporters, TcyABC and TcyJKLMN, and a symporter, TcyP (5). The TcyJKLMN and TcyP uptake systems are highaffinity transporters with apparent K m values for L-cystine of 2.5 M and 0.6 M, respectively. In addition, the TcyJKLMN system is involved in the uptake of cystathionine, S-methylcysteine, djenkolic acid, and other sulfur compounds (5, 39). The tcyJKLMN genes ...

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“…In APR from B. subtilis, on the basis of its short half-life (t1 ⁄ 2 , ϳ12 min), an oxygen-sensing function for has been proposed for the iron-sulfur cluster (22). By comparison, the iron-sulfur in MtAPR is significantly more stable with a half-life of ϳ6 h (13).…”

Section: Discussionmentioning

confidence: 99%

“…Photoreduction in the presence of deazaflavin and oxalate turned out to be the most effective method to generate the paramagnetic 1ϩ state of MtAPR. By contrast, attempts to photochemically reduce APR from plants and bacteria such as P. aeruginosa and Bacillus subtilis have not been successful (13,22). Active site residues that interact with the iron-sulfur cluster and/or the substrate (e.g.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

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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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Characterization and Reconstitution of a 4Fe-4S Adenylyl Sulfate/Phosphoadenylyl Sulfate Reductase from Bacillus subtilis

Cited by 60 publications

References 31 publications

Global Control of Cysteine Metabolism by CymR inBacillus subtilis

Global Control of Cysteine Metabolism by CymR inBacillus subtilis

Regulation of the Bacillus subtilis ytmI Operon, Involved in Sulfur Metabolism

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

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