Probing the Promiscuous Active Site of myo-Inositol Dehydrogenase Using Synthetic Substrates, Homology Modeling, and Active Site Modification

Abstract

The active site of myo-inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis recognizes a variety of mono- and disaccharides, as well as 1l-4-O-substituted inositol derivatives. It catalyzes the NAD+-dependent oxidation of the axial alcohol of these substrates with comparable kinetic constants. We have found that 4-O-p-toluenesulfonyl-myo-inositol does not act as a substrate for IDH, in contrast to structurally similar compounds such as those bearing substituted benzyl substituents in the same position. X-ray crystallographic analysis of 4-O-p-toluenesulfonyl-myo-inositol and 4-O-(2-naphthyl)methyl-myo-inositol, which is a substrate for IDH, shows a distinct difference in the preferred conformation of the aryl substituent. Conformational analysis of known substrates of IDH suggests that this conformational difference may account for the difference in reactivity of 4-O-p-toluenesulfonyl-myo-inositol in the presence of IDH. A sequence alignment of IDH with the homologous glucose-fructose oxidoreductase allowed the construction of an homology model of inositol dehydrogenase, to which NADH and 4-O-benzyl-scyllo-inosose were docked and the active site energy minimized. The active site model is consistent with all experimental results and suggests that a conserved tyrosine-glycine-tyrosine motif forms the hydrophobic pocket adjoining the site of inositol recognition. Y233F and Y235F retain activity, while Y233R and Y235R do not. A histidine-aspartate pair, H176 and D172, are proposed to act as a dyad in which H176 is the active site acid/base. The enzyme is inactivated by diethyl pyrocarbonate, and the mutants H176A and D172N show a marked loss of activity. Kinetic isotope effect experiments with D172N indicate that chemistry is rate-determining for this mutant.

Inositol Catabolism, a Key Pathway in Sinorhizobium meliloti for Competitive Host Nodulation

“…Thus, we conclude that the initial dehydrogenation of myo-as well as of D-chiro-inositol is carried out by the idhA-encoded dehydrogenase. This is not without precedent; the purified myo-inositol dehydrogenase of B. subtilis has been shown to oxidize both myo-and D-chiro-inositol (8). The S. meliloti idhA mutant was able to use scyllo-inositol as the sole C source, indicating that there is at least one other dehydrogenase involved in the oxidation of scyllo-inositol and that scyllo-inositol is probably not a substrate for the myo-inositol dehydrogenase.…”

Purification, crystallization and preliminary X-ray analysis of inositol dehydrogenase (IDH) fromBacillus subtilis

“…For molecular replacement, the programs MrBUMP (Keegan & Winn, 2007) and Phaser (McCoy et al, 2005) from the CCP4 suite were used, with search models that included structures with 15-22% sequence identity to IDH and a molecular model for the B. subtilis IDH that had recently been generated (Daniellou et al, 2007) with MODELLER (Marti-Renom et al, 2000) using GFOR from Z. mobilus as a template (PDB code 1ofg; Kingston et al, 1996;Daniellou et al, 2007). A clear molecular-replacement solution was found using a monomer of PDB entry 2glx [NADP(H)-dependent 1,5-anhydro-d-fructose reductase from Sinorhizobium morelense; 22% sequence identity to IDH] as the search model (with the cofactor and water molecules removed).…”

“…The enzyme is active as a tetramer in solution and has an apparent molecular weight of 160 kDa (Ramaley et al, 1979). Recently, an IDH model has been generated (Daniellou et al, 2007) based on the crystal structure of glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis (PDB code 1ofg; Kingston et al, 1996;Daniellou et al, 2007). Sequence alignment with IDH homologues and homology modelling of IDH allowed us to predict the active-site residues important for binding and catalysis.…”

“…His176 and Asp172 are proposed to act as the catalytic dyad, in which His176 is proposed to be the acid/base catalyst. H176A and D172N mutants showed a marked loss in activity (Daniellou et al, 2007). However, knowledge of their actual role in catalysis requires determination of the precise role of the active-site residues as provided by highresolution crystal structures with and without substrates or inhibitors.…”

Purification, crystallization and room-temperature X-ray diffraction of inositol dehydrogenase LcIDH2 fromLactobacillus caseiBL23

“…Inositol dehydrogenase (IDH) catalyzes the NAD + -dependent conversion of myo-inositol into scyllo-inosose (Ramaley et al, 1979;Daniellou et al, 2007). Many bacterial species contain multiple apparent IDH-encoding genes, although it is unknown whether these genes represent redundancies or whether they possess different substrate specificities.…”