The Presence of an Iron-Sulfur Cluster in Adenosine 5′-Phosphosulfate Reductase Separates Organisms Utilizing Adenosine 5′-Phosphosulfate and Phosphoadenosine 5′-Phosphosulfate for Sulfate Assimilation

Abstract

It was generally accepted that plants, algae, and phototrophic bacteria use adenosine 5-phosphosulfate (APS) for assimilatory sulfate reduction, whereas bacteria and fungi use phosphoadenosine 5-phosphosulfate (PAPS). The corresponding enzymes, APS and PAPS reductase, share 25-30% identical amino acids. Phylogenetic analysis of APS and PAPS reductase amino acid sequences from different organisms, which were retrieved from the GenBank TM , revealed two clusters. The first cluster comprised known PAPS reductases from enteric bacteria, cyanobacteria, and yeast. On the other hand, plant APS reductase sequences were clustered together with many bacterial ones, including those from Pseudomonas and Rhizobium. The gene for APS reductase cloned from the APS-reducing cyanobacterium Plectonema also clustered together with the plant sequences, confirming that the two classes of sequences represent PAPS and APS reductases, respectively. Compared with the PAPS reductase, all sequences of the APS reductase cluster contained two additional cysteine pairs homologous to the cysteine residues involved in binding an iron-sulfur cluster in plants. Mö ssbauer analysis revealed that the recombinant APS reductase from Pseudomonas aeruginosa contains a [4Fe-4S] cluster with the same characteristics as the plant enzyme. We conclude, therefore, that the presence of an iron-sulfur cluster determines the APS specificity of the sulfatereducing enzymes and thus separates the APS-and PAPSdependent assimilatory sulfate reduction pathways.For all living organisms, sulfur is an essential element with many different functions. It is found in reduced form in amino acids, peptides, and proteins and in iron-sulfur clusters, lipoic acid, and other cofactors and in oxidized form as sulfonate group-modifying proteins, polysaccharides, and lipids. Reduced sulfur compounds, such as hydrogen sulfide, serve as electron donors for chemotrophic or phototrophic growth in a large and diverse group of Archae and bacteria, including purple and green sulfur bacteria (1). On the other hand, oxidized sulfur compounds such as sulfate can function as a terminal electron acceptor in respiration to support the growth of sulfate-reducing bacteria (2).The majority of sulfur in living organisms is present in the reduced form of organic thiols. For their synthesis, inorganic sulfate is reduced and incorporated into bioorganic compounds in a pathway named assimilatory sulfate reduction. Before reduction, sulfate is activated with ATP to adenosine 5Ј-phosphosulfate (APS), 1 which can subsequently be converted into phosphoadenosine 5Ј-phosphosulfate (PAPS) using a second ATP. Either form of activated sulfate can be reduced to sulfite and reduced further to sulfide by sulfite reductase. Sulfide is incorporated into an activated amino acid acceptor, such as O-acetylserine, O-acetylhomoserine, or O-succinylhomoserine, to form cysteine or homocysteine (3-5).The assimilatory sulfate reduction pathway is present in plants, fungi, and yeast and in a wide range of eubacteria but is mi...

Extensive Genomic Plasticity in Pseudomonas aeruginosa Revealed by Identification and Distribution Studies of Novel Genes among Clinical Isolates

“…There are two systems for assimilatory sulfate reduction: (i) plants, algae, and phototrophic bacteria utilize adenosine 5Ј-phosphosulfate (APS), and (ii) chemotrophic bacteria and fungi use PAPS. APS reductase and PAPS reductase enzymes are key elements of their respective metabolic systems (8,26). It was shown previously that P. aeruginosa uses a plant-like APS rather than PAPS for its sulfate assimilation (5).…”

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

“…Although numerous studies indicate that the ironsulfur cluster is essential for APR activity (1,5,7,13), the specific role of the iron-sulfur cluster has been elusive. Progress on this front has been limited, in part, by the inability to generate a paramagnetic state of the [4Fe-4S] cluster that can be studied by EPR spectroscopy.…”

“…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.…”

“…Given the unusual Cys-Cys dyad coordination and its requirement for catalytic activity, defining the function and properties of the iron-sulfur cluster in APR has generated considerable interest (1,5,7,16,17). 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).…”

“…Other interactions with the iron-sulfur cluster involve Thr-87 and Trp-246. In the active site, the phosphosulfate group of APS is positioned opposite the [4Fe-4S] cluster, and although no atoms intervene, the sulfate moiety is not in direct contact with the [4Fe-4S] cluster.Given the unusual Cys-Cys dyad coordination and its requirement for catalytic activity, defining the function and properties of the iron-sulfur cluster in APR has generated considerable interest (1,5,7,16,17). 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).…”