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Crystal structure of ATP sulfurylase from Saccharomyces cerevisiae, a key enzyme in sulfate activation.

ABSTRACT: ATP sulfurylases (ATPSs) are ubiquitous enzymes that catalyse the primary step of intracellular sulfate activation: the reaction of inorganic sulfate with ATP to form adenosine-5'-phosphosulfate (APS) and pyrophosphate (PPi). With the crystal structure of ATPS from the yeast Saccharomyces cerevisiae, we have solved the first structure of a member of the ATP sulfurylase family. We have analysed the crystal structure of the native enzyme at 1.95 Angstroms resolution using multiple isomorphous replacement (MIR) and, subsequently, the ternary enzyme product complex with APS and PPi bound to the active site. The enzyme consists of six identical subunits arranged in two stacked rings in a D:3 symmetric assembly. Nucleotide binding causes significant conformational changes, which lead to a rigid body structural displacement of domains III and IV of the ATPS monomer. Despite having similar folds and active site design, examination of the active site of ATPS and comparison with known structures of related nucleotidylyl transferases reveal a novel ATP binding mode that is peculiar to ATP sulfurylases.

SUBMITTER: Ullrich TC

PROVIDER: S-EPMC133462 | biostudies-literature | 2001 Feb

REPOSITORIES: biostudies-literature

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Crystal structure of ATP sulfurylase from Saccharomyces cerevisiae, a key enzyme in sulfate activation.

Ullrich T C TC Blaesse M M Huber R R

The EMBO journal 20010201 3

ATP sulfurylases (ATPSs) are ubiquitous enzymes that catalyse the primary step of intracellular sulfate activation: the reaction of inorganic sulfate with ATP to form adenosine-5'-phosphosulfate (APS) and pyrophosphate (PPi). With the crystal structure of ATPS from the yeast Saccharomyces cerevisiae, we have solved the first structure of a member of the ATP sulfurylase family. We have analysed the crystal structure of the native enzyme at 1.95 Angstroms resolution using multiple isomorphous repl ...[more]

PMID: 11157739

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Project description:ATPS Sulfurylase (ATPS) is the first of three enzymes in the sulfate reduction pathway - one of the oldest metabolic pathways on Earth, utilized by Sulfate Reducing Bacteria (SRB). Due to the low redox potential of the sulfate ion, its reduction requires activation via formation of adenosine 5'-phosphosulfate (APS), which is catalyzed by ATPS. Dispersion-corrected hybrid density functional theory (DFT/B3LYP-D3) was used to test three reaction mechanisms proposed for conversion of ATP to APS: two-step SN-1 reaction running through AMP anhydride intermediate, two-step reaction involving cyclic AMP intermediate and direct SN-2 conversion of ATP to APS molecule. The study employed five different cluster models of the ATPS active site: one containing magnesium cation and four without it, constructed based on the crystal structure (PDB code: 1G8H) solved for ATPS from Saccharomyces cerevisiae in complex with APS and pyrophosphate (PPi), where Mg2+ was not detected. The model with magnesium ion was constructed based on the representative structure obtained from trajectory analysis of the molecular dynamics simulations (MD) performed for the hexameric ATPS-APS-Mg2+-PPi complex. The results obtained for all considered models suggest that ATPS-AMP anhydride intermediate is a highly energetic and unstable complex, while formation of cyclic AMP molecule requires formation of unfavorable hypervalent geometry at the transition state. Among all tested mechanism, the energetically most feasible mechanism of the ATPS reaction is SN-2 one-step conversion of ATP to APS occurring via a pentavalent transition state. Interestingly, such a reaction is inhibited by the presence of Mg2+ in the ATPS active site. Magnesium cation forces unfavorable geometry of reactants for SN-2 mechanism and formation of pentavalent transition state. Such a reaction requires rearrangement of Mg2+ ligands, which raises the barrier from 11-14 kcal/mol for the models without Mg2+ to 48 kcal/mol for model with magnesium ion included.

Project description:BackgroundAs the third enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase (6PGDH) is the main generator of cellular NADPH. Both thioredoxin reductase and glutathione reductase require NADPH as the electron donor to reduce oxidized thioredoxin or glutathione (GSSG). Since thioredoxin and GSH are important antioxidants, it is not surprising that 6PGDH plays a critical role in protecting cells from oxidative stress. Furthermore the activity of 6PGDH is associated with several human disorders including cancer and Alzheimer's disease. The 3D structural investigation would be very valuable in designing small molecules that target this enzyme for potential therapeutic applications.ResultsThe crystal structure of 6-phosphogluconate dehydrogenase (6PGDH/Gnd1) from Saccharomyces cerevisiae has been determined at 2.37 A resolution by molecular replacement. The overall structure of Gnd1 is a homodimer with three domains for each monomer, a Rossmann fold NADP+ binding domain, an all-alpha helical domain contributing the majority to hydrophobic interaction between the two subunits and a small C-terminal domain penetrating the other subunit. In addition, two citrate molecules occupied the 6PG binding pocket of each monomer. The intact Gnd1 had a Km of 50 +/- 9 microM for 6-phosphogluconate and of 35 +/- 6 microM for NADP+ at pH 7.5. But the truncated mutants without the C-terminal 35, 39 or 53 residues of Gnd1 completely lost their 6PGDH activity, despite remaining the homodimer in solution.ConclusionThe overall tertiary structure of Gnd1 is similar to those of 6PGDH from other species. The substrate and coenzyme binding sites are well conserved, either from the primary sequence alignment, or from the 3D structural superposition. Enzymatic activity assays suggest a sequential mechanism of catalysis, which is in agreement with previous studies. The C-terminal domain of Gnd1 functions as a hook to further tighten the dimer, but it is not necessary for the dimerization. This domain also works as a lid on the substrate binding pocket to control the binding of substrate and the release of product, so it is indispensable for the 6PGDH activity. Moreover, the co-crystallized citrate molecules, which mimic the binding mode of the substrate 6-phosphogluconate, provided us a novel strategy to design the 6PDGH inhibitors.

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Crystal structure of ATP sulfurylase from Saccharomyces cerevisiae, a key enzyme in sulfate activation.

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Crystal structure of ATP sulfurylase from Saccharomyces cerevisiae, a key enzyme in sulfate activation.

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