Crystal Structure of ATP Sulfurylase from Penicillium chrysogenum:  Insights into the Allosteric Regulation of Sulfate Assimilation^,

Abstract

ATP sulfurylase from Penicillium chrysogenum is an allosterically regulated enzyme composed of six identical 63.7 kDa subunits (573 residues). The C-terminal allosteric domain of each subunit is homologous to APS kinase. In the presence of APS, the enzyme crystallized in the orthorhombic space group (I222) with unit cell parameters of a = 135.7 A, b = 162.1 A, and c = 273.0 A. The X-ray structure at 2.8 A resolution established that the hexameric enzyme is a dimer of triads in the shape of an oblate ellipsoid 140 A diameter x 70 A. Each subunit is divided into a discreet N-terminal domain, a central catalytic domain, and a C-terminal allosteric domain. Two molecules of APS bound per subunit clearly identify the catalytic and allosteric domains. The sequence 197QXRN200 is largely responsible for anchoring the phosphosulfate group of APS at the active site of the catalytic domain. The specificity of the catalytic site for adenine nucleotides is established by specific hydrogen bonds to the protein main chain. APS was bound to the allosteric site through sequence-specific interactions with amino acid side chains that are conserved in true APS kinase. Within a given triad, the allosteric domain of one subunit interacts with the catalytic domain of another. There are also allosteric-allosteric, allosteric-N-terminal, and catalytic-catalytic domain interactions across the triad interface. The overall interactions-each subunit with four others-provide stability to the hexamer as well as a way to propagate a concerted allosteric transition. The structure presented here is believed to be the R state. A solvent channel, 15-70 A wide exists along the 3-fold axis, but substrates have access to the catalytic site only from the external medium. On the other hand, a surface "trench" links each catalytic site in one triad with an allosteric site in the other triad. This trench may be a vestigial feature of a bifunctional ("PAPS synthetase") ancestor of fungal ATP sulfurylase.

Identification and Functional Characterization of the Novel BM-motif in the Murine Phosphoadenosine Phosphosulfate (PAPS) Synthetase

“…Independent studies of bifunctional SK1 (9) and monofunctional APS kinase (16) have shown that a single mutation in the key residues of the P-loop is sufficient to inactivate the kinase. Loss of kinase activity in our D87A mutant supports the hypothesis put forward by MacRae et al (15,16) that lack of APS kinase activity in the ATP sulfurylase regulatory domain is due both to the altered P-loop and the absence of an aspartate at residue 432. Additional conserved residues, C-terminal to the DGD-turn, also contribute to PAPS synthetase activity.…”

“…However, structural comparison of P. chrysogenum APS kinase with substrate-bound structures of guanylate kinase (13) and 6-phosophofructo-2-kinase (14) suggest that APS is placed between the P-loop, FISP-motif, and the ␣-helix C-terminal of the DGD-loop. Furthermore, during the course of our functional analysis of the BM-motif, MacRae et al crystallized P. chrysogenum ATP sulfurylase in the presence of APS (15), and showed that APS was bound to the sulfurylase active site as well as to the C-terminal kinase-like regulatory domain. As we predicted, this crystal structure shows residue Asp 434 , analogous to Asp 89 in SK1, interacting with the 3Ј-OH of APS, thereby aiding in proper alignment of APS for phosphoryl transfer.…”

Structure and Mechanism of Soybean ATP Sulfurylase and the Committed Step in Plant Sulfur Assimilation

“…These studies also reveal the versatility of the ATP sulfurylase fold as sequence variations that alter the oligomerization interface lead to a range of architectures for this enzyme (Figs. 3 and 4) (43)(44)(45)(47)(48)(49)(50)(51).…”

“…Of the ATP sulfurylases structurally related to GmATPS (Figs. 1 and 4), only the P. chrysogenum enzyme has a demonstrated allosteric regulatory mechanism involving PAPS (49,50). The observed structural transition in response to PAPS levels provides for control of sulfate activation in P. chrysogenum, but it does not appear to be a widely used regulatory feature in other organisms.…”

“…In other prokaryotes, including the thermophiles Aquifex aeolicus and Thermus thermophilus, the purple sulfur bacterium Allochromatium vinosum, and a bacterial symbiont of the vent tubeworm Riftia pachyptila, ATP sulfurylase functions either as a monofunctional homodimeric enzyme or a bi-functional homodimeric ATP sulfurylase/APS kinase, also known as PAPS synthetase (43)(44)(45)(46). In fungi, such as Saccharomyces cerevisiae and Penicillium chrysogenum, ATP sulfurylase is a homohexamer in which each monomer contains an N-terminal ATP sulfurylase domain and a C-terminal APS kinase-like domain (47)(48)(49). The APS kinase-like domain can also function as an allosteric regulatory region in response to PAPS, as shown for the P. chrysogenum enzyme (50).…”