The Structure of the 1l-myo-inositol-1-phosphate Synthase-NAD+-2-deoxy-d-glucitol 6-(E)-Vinylhomophosphonate Complex Demands a Revision of the Enzyme Mechanism

Abstract

1L-myo-inositol 1-phosphate (MIP) synthase catalyzes the conversion of D-glucose 6-phosphate to 1L-myo-inositol 1-phosphate, the first and rate-limiting step in the biosynthesis of all inositol-containing compounds. It involves an oxidation, enolization, intramolecular aldol cyclization, and reduction. Here we present the structure of MIP synthase in complex with NAD ؉ and a high-affinity inhibitor, 2-deoxy-D-glucitol 6-(E)-vinylhomophosphonate. This structure reveals interactions between the enzyme active site residues and the inhibitor that are significantly different from that proposed for 2-deoxy-Dglucitol 6-phosphate in the previously published structure of MIP synthase-NAD ؉ -2-deoxy-D-glucitol 6-phosphate. There are several other conformational changes in NAD ؉ and the enzyme active site as well. Based on the new structural data, we propose a new and completely different mechanism for MIP synthase.Inositol-containing compounds play critical and diverse biological roles, including signal transduction, stress response, and cell wall biogenesis (1-4). Though large quantities of inositol are available from the diet, significant biosynthesis of inositol has been detected in organs where a significant blood barrier exists, such as the testes and brain (5-9). In fact, reduction of the brain inositol pool by inhibition of myo-inositol (MI) 1 monophosphatase has been suggested to be the mode of action for lithium in the treatment of bipolar disorder (10 -13). Recent in vivo results in yeast (14) and dictyostelium (15) suggest that Valproate, a drug used in the treatment of depression, bipolar disorder, and seizure disorder, may act by inhibition of MIP synthase, thus lowering neuronal inositol pools similar to the action of lithium (14). Regulation of inositol biosynthesis itself may, therefore, play an important role in the regulation of second messenger signaling.MIP synthase is remarkably conserved in eukaryotes, with better than 45% identity from yeast to humans (3, 16 -25). In all cases, the enzyme displays modest catalytic activity, with turnover numbers ranging from 3 to 13 M/min/mg enzyme and with substrate K m values in the 100 M to 1 mM range (3, 17). The reaction path first proposed by Loewus et al. (shown in Fig. 1) remains the most likely (3, 26 -29). A series of inhibitor studies indicates that the enzyme first binds the open acyclic tautomer of the substrate D-glucose 6-phosphate, followed by oxidation to the C5 keto intermediate (30). Frost and coworkers propose that enolization is promoted by proton extraction by means of the substrate phosphate, which is consistent with the phosphate binding in a transoid conformation (30). They base this conclusion on the ability of 2-deoxy-D-glucitol 6-(E)-vinylhomophosphonate, a substrate mimic of the enzyme that fixes the phosphonate trans to C5, to strongly inhibit the enzyme, whereas the equivalent Z-mimic displays no affinity for the enzyme. 2-Deoxy-D-glucitol 6-(E)-vinylhomophosphonate is, in fact, the most potent inhibitor of the enzyme known, having ...

Phosphorylation Regulates myo-Inositol-3-phosphate Synthase

“…The adenine portion of NAD ϩ specifically forms tight hydrogen bonds between N1 and Ser-184 (16). Structural studies suggest that the binding of NAD ϩ to the apoenzyme is a prerequisite for the orderly binding of the substrate glucose 6-phosphate to the active site (16,38). Therefore, it is likely that NAD ϩ binding is disrupted when Ser-184 is phosphorylated, resulting in decreased activity of MIPS, which is dependent on NAD ϩ as a cofactor.…”

Probing the Mechanism of the Archaeoglobus fulgidus Inositol-1-phosphate Synthase

“…Although one could explain decreased activity for both these mutants, what is more surprising is the dramatically reduced binding of G-6-P to each enzyme, whereas P i still binds to both. By analogy to the yeast enzyme (14,16,17), Asn 255 could interact with the 2-OH of linearized G-6-P (although it is further away in the A. fulgidus enzyme), and Lys 367 would be necessary for orienting the 5-hydroxy of G-6-P (or the keto group in 5-keto-G-6-P). There are several other residues in the active site that could hydrogen bond to acyclic G-6-P.…”

“…The yeast enzyme is the only one with a substrate analog bound, and it is oriented in an extended conformation that does not have substrate C-1 or C-6 near one another (17). Inhibitors of the enzyme that are analogs of acyclic G-6-P bind quite tightly and are potent inhibitors of the yeast mIPS, whereas cyclic compounds are very poor inhibitors (19).…”

“…Additional evidence for significant conformational changes along the pathway and sequestration of the intermediates from dissociation into solution are needed to draw more detailed conclusions about the role of a given residue in catalysis. Indeed, several active site mutants of yeast and M. tuberculosis mIPS have been generated based on the crystal structures (17,18) and shown to be inactive. Exactly at what point in the mechanism the activity has been impaired was difficult to deduce from those experiments.…”

“…NAD ϩ binding to vacant sites decreases the intrinsic fluorescence intensity but not the wavelength of maximum emission of the archaeal mIPS protein. This change in fluorescence intensity can be used to quantify NAD ϩ binding to the proteins by measuring the intrinsic fluorescence intensity at 334 nm compared with that for the protein where only buffer was added over an NAD ϩ concentration range of [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] M. There are two tryptophans near the active site of A. fulgidus mIPS (13) that could have altered characteristics upon NAD ϩ binding. From the change in fluorescence intensity as a function of added NAD ϩ , we used the concentration for 50% of the maximum change to define an apparent K D for NAD ϩ binding (see Fig.…”