L-myo-inositol 1-phosphate synthase (MIPS; EC 5.5.1.4) catalyzes the rate-limiting step in the synthesis of myo-inositol, a critical compound in the cell. Plants contain multiple MIPS genes, which encode highly similar enzymes. We characterized the expression patterns of the three MIPS genes in Arabidopsis thaliana and found that MIPS1 is expressed in most cell types and developmental stages, while MIPS2 and MIPS3 are mainly restricted to vascular or related tissues. MIPS1, but not MIPS2 or MIPS3, is required for seed development, for physiological responses to salt and abscisic acid, and to suppress cell death. Specifically, a loss in MIPS1 resulted in smaller plants with curly leaves and spontaneous production of lesions. The mips1 mutants have lower myo-inositol, ascorbic acid, and phosphatidylinositol levels, while basal levels of inositol (1,4,5)P 3 are not altered in mips1 mutants. Furthermore, mips1 mutants exhibited elevated levels of ceramides, sphingolipid precursors associated with cell death, and were complemented by a MIPS1-green fluorescent protein (GFP) fusion construct. MIPS1-, MIPS2-, and MIPS3-GFP each localized to the cytoplasm. Thus, MIPS1 has a significant impact on myo-inositol levels that is critical for maintaining levels of ascorbic acid, phosphatidylinositol, and ceramides that regulate growth, development, and cell death.

UDP-3-O-((R)

No abstract

2 as their primary reducing agent and in xenobiotic metabolism for the detoxification of drugs and other toxins (1-4). MSH is likely to be critical for the survival of mycobacteria inside activated macrophages, where the mycobacteria are subjected to oxidative bursts. Consequently, the enzymes involved in MSH biosynthesis and detoxification (Fig. 1A), including the metalloenzymes N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-␣-D-glucopyranoside deacetylase (MshB) and MSH-conjugate amidase, are targets for the development of antibiotics for the treatment of diseases such as tuberculosis (5-10).The enzyme MshB catalyzes the hydrolysis of (GlcN-Ins) and acetate, the fourth overall step in MSH biosynthesis (rate-limiting step) (11). MshB is an attractive drug target because it is a metalloenzyme; there are past successes in targeting metalloenzymes, including inhibitors of carbonic anhydrase, matrix metalloproteases, and angiotensinconverting enzyme (12-15). Inhibitors of metalloenzymes typically contain a group that binds to the catalytic metal ion. Consequently, a comprehensive understanding of metalloenzyme cofactor preferences is necessary for the development of potent and specific metalloenzyme inhibitors.MshB was previously identified as a Zn 2ϩ -dependent enzyme based on the observations that the enzyme copurifies with Zn 2ϩ (Fig. 1B) and that the enzyme activity is reversibly inhibited by treatment with 1,10-phenanthroline (16 -18). On the basis of the structure of the enzyme active site, MshB is thought to catalyze the hydrolysis of GlcNAc-Ins via one of two potential chemical mechanisms using general acid-base catalysis (GABC) (19). One possible mechanism uses a single bifunctional GABC to facilitate the hydrolysis of GlcNAc-Ins, whereas the other uses a GABC pair to carry out this reaction. However, Fe 2ϩ was not examined as a potential cofactor in these experiments. Furthermore, MshB was purified using zinc immobilized metal ion affinity chromatography (IMAC) under aerobic conditions, which is biased toward zinc incorporation into metalloenzymes (16). Purified MshB contains nickel (0.82 eq) when purified using nickel IMAC (aerobic conditions) (16). There have been several examples over the last decade of Fe 2ϩ -enzymes being misidentified as exclusive Zn 2ϩ -enzymes, including peptide deformylase, S-ribosylhomocysteinase (LuxS), UDP-3-O-(R-3-hydroxymyristoyl)-Nacetylglucosamine deacetylase (LpxC), and possibly histone deacetylase-8 (HDAC8) (20 -27). In all these enzymes, the Fe 2ϩ cofactor is either exclusively preferred or is preferred over Zn 2ϩ under certain environmental conditions. The

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a metal-dependent deacetylase that catalyzes the hydrolysis of UDP-3-O-myristoyl-N-acetyl-glucosamine to form UDP-3-O-myristoyl-glucosamine and acetate. This is the committed step in the biosynthesis of lipid A, and therefore, LpxC is a target for the development of antimicrobial agents in the treatment of Gram-negative infections. To facilitate the development of potent and specific inhibitors of LpxC, the molecular determinants of binding and specificity and the catalytic mechanism for this enzyme have been probed. The functions of active site residues have been classified on the basis of changes in steady-state turnover (kcat, KM, and kcat/KM) and product binding affinity (KDProduct). We have identified side chains that enhance product affinity and reactivity (F192, K239, D246, and H265), destabilize product affinity (E78 and D197), and preferentially enhance catalytic efficiency (H19, T19, K143, and N162). In addition, the affinity of LpxC for myrUDP-GlcNH2 is dependent on two ionizations, one deprotonation and one protonation, with apparent pKa values of 6.5 +/- 0.1 and 7.4 +/- 0.1, respectively. The UDP moiety of the product contributes significantly to recognition by LpxC, suggesting that this region can be targeted in drug development. These data provide a map of the active site features essential for catalysis and molecular recognition by LpxC that can be used for developing more potent LpxC inhibitors.