Cloning and sequencing of ATP sulfurylase from Penicillium chrysogenum: Identification of a likely allosteric domain

Section of Molecular and Cellular Biology, University of California, Davis 95616.
Journal of Biological Chemistry (Impact Factor: 4.57). 09/1994; 269(31):19777-86.
Source: PubMed


Fungal (Penicillium chrysogenum) and yeast (Saccharomyces cerevisiae) ATP sulfurylases were shown to have very similar kinetic and chemical properties except that the fungal enzyme (a) contains a highly reactive Cys residue (SH-1) whose modification results in sigmoidal velocity curves (Renosto, F., Martin, R. L., and Segel, I. H. (1987) J. Biol. Chem. 262, 16279-16288) and (b) is allosterically inhibited by 3′-phosphoadenosine 5′-phosphosulfate (PAPS), while the yeast enzyme displays neither of these properties. The fungal enzyme subunit (64.3 kDa, 572 amino acids) is also larger than the yeast enzyme subunit (59.3 kDa, 521 amino acids). To correlate the unique allosteric properties of the fungal enzyme with specific structural features, we cloned and sequenced the ATP sulfurylase gene (aps) from P. chrysogenum. The yeast and fungal enzymes are homologous over the first 400 amino acids and contain two regions high in basic residues which are conserved in sulfurylases from Arabidopsis and the Riftia pachyptila (hydrothermal vent tube worm) chemolithotrophic symbiont. These regions may participate in forming the binding sites for MgATP2- and SO42-. The fungal enzyme has no significant sequence homology to the yeast enzyme in the C-terminal 172 amino acids. This C-terminal region contains SH-1 (Cys-508) and has homology to MET14 (S. cerevisiae), CYSC (E. coli), and NODQ (Rhizobium meliloti), i.e. adenosine 5′-phosphosulfate (APS) kinase. The cumulative results suggest that (a) the allosteric PAPS binding site of P. chrysogenum ATP sulfurylase is located in the C-terminal domain of the protein and (b) that this domain may have evolved from APS kinase. In spite of the homology, this C-terminal region does not account for the APS kinase activity of P. chrysogenum. Fungal ATP sulfurylase has no significant homology to (or regulatory properties in common with) CYSD or CYSN, proteins reported to comprise E. coli ATP sulfurylase.

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    • "The GTPase subunit CysN activates the catalytic subunit CysD by allosteric interactions (Wei et al., 2000). ATPS from sulfate-reducing bacteria and eukaryotic organisms such as yeast (Karamohamed et al., 1999), ®lamentous fungi (Foster et al., 1994) and plants (Leustek et al., 1994; Logan et al., 1996) have been described as monomers or homo-oligomeric complexes, which do not require GTP for activation. However, in higher eukaryotes, like the marine worm (Rosenthal and Leustek, 1995) and mammalian species (Li et al., 1995; Venkatachalam et al., 1998), gene fusion has occurred; ATPS and the APS kinase, which performs the ®nal step of the synthesis of the highenergy sulfate donor PAPS (3¢-phosphodenosine- 5¢-phosphosulfate) by 3¢-phosphorylation of APS, are arranged as subdomains on a single polypeptide chain. "
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    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.
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    • "The sulphate permease gene(s) of A. nidulans and P. chrysogenum has (ve) yet to be isolated, and would appear to be the most likely targets of the transcriptional regulation of sulphate assimilation by methionine, as in N. crassa. However, there is also some evidence from kinetic studies on purified P. chrysogenum ATP sulphurylase playing a role in the allosteric control of the pathway by the action of PAPS on this enzyme, perhaps involving the the C-terminal, APS kinase-like region (Foster et al. 1994). "
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