ATP-sulfurylase

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Introduction

Sulfate Reducing Bacteria (SRB) participate in one of the oldest enzymatic pathways on Earth: the dissimilatory reduction of sulfate (SO4^2-) to sulfide (S^2-). Among SRB, some bacteria and archaea can use sulfate as a terminal electron acceptor during their metabolism under aerobic conditions. Sulfite (SO3^2-) acts as an intermediate to mediate the reduction process of sulfate. SRB plays an important role in the biogeochemical sulfur cycle and the biodegradation of aromatic pollutants, and can be used as a regulator of important processes in soils (such as mercury methylation, etc.).

The pathway of dissimilative reduction of sulfate to anionic sulfide involves three cytoplasmic enzymes: ATP Sulfurylase (ATPS), adenosine 5′-phosphosulfate (APS) Reductase (APSR) and dissimilatory sulfite Reductase (dSIR). Among them, ATPS is responsible for the formation of SO4^2--AMP bond to catalyze the activation of sulfate anion to adenosine 5′-phosphosulfate (APS). The reason why this kind of SO4^2- activation is required is because it has a very low redox potential and cannot be directly reduced by organic acids or molecular hydrogen. Sulfide released through this metabolic pathway may cause corrosion of the metallic construction in the living environment of SRB. In the assimilation pathway of eukaryotes, certain bacteria and algae, the APS product of the ATPS reaction is the substrate for APS kinase, which phosphorylates at 3'-position to produce 3′-phosphoadenosine5′-phosphosulfate (PAPS), PAPS acts as a sulphuryl donor for many sulfur-containing compounds. It may also be reduced to sulfide needed for biosynthesis of cysteine, which may be the sulfur donor of methionine. In metazoa, the ATPS and APS kinases of fungi and certain algae gather in a protein called PAPS synthase. Various ATPS isoforms have been found in microalgae, and the diversity of ATPS isoforms in seaweed and freshwater algae suggests that the life in different sulfate availability may affect the evolution of ATPS enzymes.

Crystal structure of ATP sulfurylase

ATPS is a ubiquitous enzyme in nature that uses ATP to catalyze the reversible reaction of sulfate activation to produce APS and pyrophosphate anions. ATPS from SRB, yeast and plants are all characterized as monomeric or homo-oligomeric structures. The structures of ATPS-APS complex are available for the following organisms: soybean (PDB code: 4MAF), Penicillium chrysogenum (PDB code: 1I2D), Saccharomyces cerevisiae (PDB codes: 1G8H, 1G8G, 1JEE) and Thermus thermophillus (PDB code: 1V47). Similar to Penicillium chrysogenum (PDB code: 1I2D), the quaternary structure of ATPS from Saccharomyces cerevisiae (PDB code: 1G8H) is determined to be a homohexameric assembly arranged into two trimeric rings, which stacked with a twist of 60° (Figure 1). In the crystal structure of ATP sulfurylase, four conserved motifs are observed in its active site: QXRN (Gln195-Thr196-Arg197-Asn198), HXXH (His201-Arg202-Ala203-His204), VGRDHAG (Val288- Gly289-Arg290-Asp291-His292-Ala293-Gly294) and PFR (Pro327-Phe328-Arg329).

Figure 1. Hexamer structure of ATPS-APS-Mg2+-pyrophosphate complex obtained after 20 ns of Molecular Dynamics (MD) simulations; B. Four conserved motifs observed in the known. ATPS sequences presented in the 1G8H crystal structure (Wójcik-Augustyn, A.; et al. 2019)

Hypothesis of ATPS catalytic reaction

The crystallographic study of ATPS from Saccharomyces cerevisiae and Penicillium chrysogenum revealed that ATP binding can cause significant displacement in the hexameric ATPS structure. It has been found that ATPS is an allosteric enzyme that can adopt at least two conformations: R state with higher affinity for the substrate and T state, which is preferably allosteric inhibitors. ATP binding causes Phe328 side chain to displace, and ATPS transforms into R state. The folding of domain II containing the active site region indicates that ATPS may belong to the superfamily of α/β phosphodiesterases, but the architecture of the protomer and independent topologies present in one protein chain may also indicate that ATPS can represent a new α/β protein class. Based on the isotope trapping, mass spectrometry and kinetic studies of ATPS of Escherichia coli K12, a hypothesis of how ATPS catalyzes the reaction is put forward. The ATPS of E. coli is a tetramer composed of heterodimers encoded by cysD and cysN genes. Isotope trapping experiments show that the ATPS reaction in E. coli is a GTP-dependent two-step process. Firstly, the enzyme-AMP anhydride and pyrophosphate are formed, and then after the dissociation of pyrophosphate, sulfate anion (SO4^2-) enters the active site and forms APS. However, it is worth mentioning that the production of AMP intermediates is GTP-dependent and is inhibited by the addition of sulfate. The horseshoe-shaped conformation of ATP molecules modeled by SYBYL based on the ATPS-APS-PPi crystal structure from Saccharomyces cerevisiae is likely to cause in-line nucleophilic attack of the sulfate molecules.

Figure 2. The two-step SN-1 reaction mechanism proposed for ATPS running through AMP anhydride intermediate (Wójcik-Augustyn, A.; et al. 2019)

Figure 3. The one-step SN-2 reaction mechanism proposed for ATPS based on the modeled ATPS-ATP-sulfate structure (Wójcik-Augustyn, A.; et al. 2019)