Site-selected Mutagenesis of a Conserved Nucleotide Binding HXGH Motif Located in the ATP Sulfurylase Domain of Human Bifunctional 3′-Phosphoadenosine 5′-Phosphosulfate Synthase

Abstract

3-Phosphoadenosine-5-phosphosulfate (PAPS) synthase is a bifunctional protein consisting of an NH 2 -terminal APS kinase and a COOH-terminal ATP sulfurylase. Both catalytic activities require ATP; the APS kinase domain involves cleavage of the ␤-␥ phosphodiester bond of ATP, whereas the ATP sulfurylase domain involves cleavage of the ␣-␤ phosphodiester bond of ATP. Previous mutational studies have suggested that ␤-␥ phosphodiesterase activity involves a highly conserved NTP-binding P-loop motif located in the adenosine-5-phosphosulfate kinase domain of PAPS synthases. Sequence alignment analysis of PAPS synthases and the superfamily of TagD-related nucleotidylyltransferases revealed the presence of a highly conserved HXGH motif in the ATP sulfurylase domain of PAPS synthases, a motif implicated in the ␣-␤ phosphodiesterase activity of cytidylyltransferases. Thus, site-selected mutagenesis of the HXGH motif in the ATP sulfurylase domain of human PAPS synthase (amino acids 425-428) was performed to examine this possibility. Either H425A or H428A mutation produced an inactive enzyme. In contrast, a N426K mutation resulted in increased enzymatic activity. A G427A single mutant resulted in only a modest 30% reduction in catalytic activity, whereas a G427A/H428A double mutant produced an inactive enzyme. These results suggest an important role for the HXGH histidines in the ATP sulfurylase activity of bifunctional PAPS synthase and support the hypothesis that the highly conserved HXGH motif found in the ATP sulfurylase domain of PAPS synthases is involved in ATP binding and ␣-␤ phosphodiesterase activity.In the course of sulfonation, inorganic sulfate must be activated prior to being transferred to an acceptor molecule (1), and the activated sulfate molecule is 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS).1 The activation of inorganic sulfate to form PAPS results from the concerted action of two enzymes (1). The first step is catalyzed by ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) and involves the reaction of inorganic sulfate with ATP to form adenosine 5Ј-phosphosulfate (APS) and inorganic pyrophosphate. This reaction results in the formation of a high energy phosphoric-sulfuric acid anhydride bond that is the chemical basis for sulfate activation (2). The second step involves APS reacting with another molecule of ATP to form PAPS and ADP and is catalyzed by APS kinase (ATP:adenylylsulfate 3Ј-phosphotransferase, EC 2.7.1.25). ATP sulfurylase and APS kinase cloned from bacteria, Fungi, yeast, and plants are found on separate polypeptide chains; however, in mammalian species, the marine worm, and the fruit fly, gene fusion has occurred, and the two enzymes are integral to bifunctional PAPS synthase. It has been clearly established that the NH 2 -terminal region of human (h) PAPS synthase constitutes the APS kinase domain, whereas the ATP sulfurylase domain is located in the COOH-terminal portion of this bifunctional protein (3). That is, the NH 2 -terminal 268 amino acids of hPAPS synthase (623 amino...

A Novel Adenylate Binding Site Confers Phosphopantetheine Adenylyltransferase Interactions with Coenzyme A

“…PPAT is a member of the nucleotidyltransferase ␣/␤ phosphodiesterase superfamily (2), which includes class I aminoacyl-tRNA synthetases (7,9,27), adenylylsulfate-phosphate adenylyltransferase (6,34), glycerol-3-phosphate cytidylyltransferase (26,35), nicotinamide mononucleotide adenylyltransferase (8), and pantothenate synthetase (33). This family is characterized by the presence of a mononucleotide binding fold and a conserved T/HXGH sequence motif, with a specific catalytic role for the second histidine within this motif.…”

Chemical Modification and Site-directed Mutagenesis of Conserved HXXH and PP-loop Motif Arginines and Histidines in the Murine Bifunctional ATP Sulfurylase/Adenosine 5′-Phosphosulfate Kinase

“…Many of these conserved residues are present in motifs that have been implicated in ATP binding (P-loop) (13), phosphoryl transfer (FISP) (14), phosphodiester-cleavage (15), and pyrophosphate binding (PPloop) (16). Recently, we reported on the generation of several site-directed mutants in the ATP-binding motif of the kinase domain (17), as well as the expression of truncated, monofunctional and rearranged domains of the bifunctional enzyme (18).…”

“…Evidence supporting a putative role for arginines and histidines in the ATP sulfurylase/APS kinase reaction mechanism comes from studies of both yeast and fungal ATP sulfurylases, which are inactivated by phenylglyoxal, which modifies arginines, and by diethylpyrocarbonate, which targets histidines (32). Finally, in order to assess the putative roles of these motifs in MSK1, overall assays alone, as were recently reported for human SK1 (15), are necessary but not sufficient. Since PAPS synthetase is a bifunctional enzyme with at least two reactive centers and multiple binding sites, several factors could lead to loss of overall activity, including loss of kinase activity, loss of sulfurylase activity, or inefficient transfer of APS from sulfurylase to kinase.…”

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

“…The recently determined crystal structure of an apoenzyme form of the enzyme from the purple sulfur bacterium A. vinosum reveals movement of ␣12 to open the active site (46). Although multiple ATP sulfurylases from different organisms have been crystallized and structurally characterized, functional analysis of these enzymes is limited to the two histidines near the active site that correspond to His-252 and His-255 of GmATPS (63,64). The HXXH motif was originally identified in nucleotidyltransferases and type I amino acyltransferases (65)(66)(67).…”

“…In these enzymes, the conserved histidines are directly involved in cleavage of the ␣/␤-phosphate bond. Mutagenesis of the HXXH motif in the ATP sulfurylase domain of human and mouse PAPS synthetase led to reduced specific activity (63,64), but more detailed steady-state kinetic analysis of the effect on function was not performed. To date, no structure-function analysis of other active site residues of an ATP sulfurylase from any other species has been reported.…”