Crystal Structure of a Dual Activity IMPase/FBPase (AF2372) from Archaeoglobus fulgidus

Abstract

Several hyperthermophilic organisms contain an unusual phosphatase that has dual activity toward inositol monophosphates and fructose 1,6-bisphosphate. The structure of the second member of this family, an FBPase/IMPase from Archaeoglobus fulgidus (AF2372), has been solved. This enzyme shares many kinetic and structural similarities with that of a previously solved enzyme from Methanococcus jannaschii (MJ0109). It also shows some kinetic differences in divalent metal ion binding as well as structural variations at the dimer interface that correlate with decreased thermal stability. The availability of different crystal forms allowed us to investigate the effect of the presence of ligands on the conformation of a mobile catalytic loop independently of the crystal packing. This conformational variability in AF2372 is compared with that observed in other members of this structural family that are sensitive or insensitive to submillimolar concentrations of Li ؉ . This analysis provides support for the previously proposed mechanism of catalysis involving three metal ions. A direct correlation of the loop conformation with strength of Li ؉ inhibition provides a useful system of classification for this extended family of enzymes.In mammalian cells, inositol monophosphatases are abundant cytosolic enzymes necessary to regenerate the supply of myo-inositol for the synthesis of phosphatidylinositols. However, in archaeal hyperthermophilic organisms, e.g. Archaeoglobus fulgidus, an IMPase 1 ortholog is thought to be used in the biosynthesis of a unique osmolyte, di-myo-inositol 1,1-phosphate (1). The archaeal enzymes have another twist as well.Besides catalyzing the hydrolysis of Ins-1-P (needed in response to thermal and salt stress) they also very specifically dephosphorylate FBP at C-1 (2). Enzymes with these characteristics exist as two distinct gene products in mammalian systems and several bacteria (3). The dual specificity of the enzyme from primitive hyperthermophiles strongly suggest that IMPase and FBPase enzymes may have evolved from the same gene product (2). Structural features of the IMPase/FBPase (MJ0109) from Methanococcus jannaschii (2) are similar to those of mammalian IMPase, inositol polyphosphase phosphatase (IPPase), FBPase, and the yeast enzyme Hal2. These enzymes form an extended family for which the general principles of chemical reactivity are the same (3). Two or three metal ions are necessary for catalysis to occur. From the structure of human IMPase with bound Ins-1-P and Gd 3ϩ , it was inferred that two metal ions were needed for catalysis (4). However, crystallographic data for pig kidney FBPase (5) and for MJ0109 (6) are consistent with a three-metal ion-assisted catalytic mechanism.There is strong biochemical evidence that Li ϩ inhibits IMPase in mammalian cells where a dose response to this ion correlates with a decrease in the intracellular pool of myoinositol (7). Li ϩ , by inhibiting mammalian IMPases, is thought to cause a depletion of inositol that attenuates the synthesis of phosphat...

Structural and Biochemical Characterization of the Type II Fructose-1,6-bisphosphatase GlpX from Escherichia coli

“…Implications for the Possible Catalytic Mechanism of the Type II FBPases-The catalytic mechanism of the lithium-sensitive phosphatases has been the subject of discussion for several years (3,10,20,54,56). The phosphatase reaction of Li ϩ -sensitive phosphatases has an absolute requirement for metal ions and has been suggested to proceed through the activation of a water molecule and its subsequent nucleophilic attack on the removable phosphorus atom (57,58).…”

“…1). Therefore, like the dual specificity IMPase/FBPase enzymes AF2372 and MJ0109 (20,54), the E. coli class II FBPases belong to the group of more resistant Li ϩ -sensitive phosphatases. In addition, FBPase activity of both GlpX and YggF was inhibited by low concentrations of inorganic phosphate (IC 50 3.0 and 1.2 mM, respectively) (supplemental Fig.…”

“…Three-dimensional structures of the type I (from pig kidney, spinach chloroplasts, and E. coli), type IV (MJ0109 and AF2372), and type V (ST0318) FBPases have been solved (10,11,19,20,22,23). FBPases I and IV and inositol monophosphatases share a common sugar phosphatase fold organized in five layered interleaved ␣-helices and ␤-sheets (␣-␤-␣-␤-␣) (2,19,24).…”

“…The type I FBPase is the most widely distributed among living organisms and is the primary FBPase in Esche-richia coli, most bacteria, a few archaea, and all eukaryotes (9,(11)(12)(13)(14)(15). The type II FBPases are represented by the E. coli GlpX and FBPase F-I from Synechocystis PCC6803 (9,16); type III is represented by the Bacillus subtilis FBPase (17); type IV is represented by the dual activity FBPases/inosine monophosphatases FbpA from Pyrococcus furiosus (18), MJ0109 from Methanococcus jannaschii (19), and AF2372 from Archaeoglobus fulgidus (20); and type V is represented by the FBPases TK2164 from Pyrococcus (Thermococcus) kodakaraensis and ST0318 from Sulfolobus tokodai (10,21).…”

Genomic identification and in vitro reconstitution of a complete biosynthetic pathway for the osmolyte di- myo -inositol-phosphate

“…Both enzymes are broadly conserved, and examples of each have been well characterized biochemically and structurally, including IMP and IPS from the hyperthermophiles Methanocaldococcus jannaschii (14), Archaeoglobus fulgidus (15)(16)(17)(18)(19), T. maritima (20), and Thermococcus kodakaraensis (21).…”