Regulatory Mutations of Inositol Biosynthesis in Yeast: Isolation of Inositol-Excreting Mutants

The enzyme inositol-1-phosphate synthase (I-1-P synthase), product of the INO1 locus, catalyzes the synthesis of inositol-1-phosphate from the substrate glucose-6-phosphate. The activity of this enzyme is dramatically repressed in the presence of inositol. By selecting for mutants which overproduce and excrete inositol, we have identified mutants constitutive for inositol-1-phosphate synthase as well as a mutation in phospholipid biosynthesis. Genetic analysis of the mutants indicates that at least three loci (designated OPI1, OPI2 and OPI4) direct inositol-mediated repression of I-1-P synthase. Mutants of these loci synthesize I-1-P synthase constitutively. Three loci are unlinked to each other and to INO1, the structural gene for the enzyme. A mutant of a fourth locus, OPI3, does not synthesize I-1-P synthase constitutively, despite its inositol excretion phenotype. This mutant is preliminarily identified as having a defect in phospholipid synthesis.     

Purification and biochemical characterization of Mycobacterium tuberculosis SuhB, an inositol monophosphatase involved in inositol biosynthesis

Phosphatidylinositol is an essential component of mycobacteria, and phosphatidylinositol-based lipids such as phosphatidylinositolmannosides, lipomannan, and lipoarabinomannan are major immunomodulatory components of the Mycobacterium tuberculosis cell wall. Inositol monophosphatase (EC 3.1.3.25) is a crucial enzyme in the biosynthesis of free myo-inositol from inositol-1-phosphate, a key substrate for the phosphatidylinositol synthase in mycobacteria. Analysis of the M. tuberculosis genome suggested the presence of four M. tuberculosis gene products that exhibit an inositol monophosphatase signature. In the present report, we have focused on SuhB, which possesses the highest degree of homology with human inositol monophosphatase. SuhB gene was cloned into an E. coli expression vector to over-produce a His-tagged protein, which was purified and characterized. SuhB required divalent metal ions for functional inositol monophosphatase activity, with Mg(2+) being the strongest activator. Inositol monophosphatase activity catalyzed by SuhB was inhibited by the monovalent cation lithium (IC(50) = 0.9 mM). As anticipated, inositol-1-phosphate was the preferred substrate (K(m) = 0.177 +/- 0.025 mM; k(cat) = 3.6 +/- 0.2 s(-)(1)); however, SuhB was also able to hydrolyze a variety of polyol phosphates such as glucitol-6-phosphate, glycerol-2-phosphate, and 2'-AMP. To provide further insight into the structure-function relationship of SuhB, different mutant proteins were generated (E83D, D104N, D107N, W234L, and D235N). These mutations almost completely abrogated inositol monophosphatase activity, thus underlining the importance of these residues in inositol-1-phosphate dephosphorylation. We also identified L81 as a key residue involved in sensitivity to lithium. The L81A mutation rendered SuhB inositol monophosphatase activity 10-fold more resistant to inhibition by lithium (IC(50) = 10 mM). These studies provide the first steps in the delineation of the biosynthesis of the key metabolite inositol in M. tuberculosis.     

Antisense downregulation of SARS-CoV gene expression in Vero E6 cells

BACKGROUND: Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus (SARS-CoV). It is an enveloped, single-stranded, plus-sense RNA virus with a genome of approximately 30 kb. The structural proteins E, M and N of SARS-CoV play important roles during host cell entry and viral morphogenesis and release. Therefore, we have studied whether expression of these structural proteins can be down-regulated using an antisense technique. METHODS: Vero E6 cells were transfected with plasmid constructs containing exons of the SARS-CoV structural protein E, M or N genes or their exons in frame with the reporter protein EGFP. The transfected cell cultures were treated with antisense phosphorothioated oligonucleotides (antisense PS-ODN, 20mer) or a control oligonucleotide by addition to the culture medium. RESULTS: Among a total of 26 antisense PS-ODNs targeting E, M and N genes, we obtained six antisense PS-ODNs which could sequence-specifically reduce target genes expression by over 90% at the concentration of 50 microM in the cell culture medium tested by RT-PCR. The antisense effect was further proved by down-regulating the expression of the fusion proteins containing the structural proteins E, M or N in frame with the reporter protein EGFP. In Vero E6 cells, the antisense effect was dependent on the concentrations of the antisense PS-ODNs in a range of 0-10 microM or 0-30 microM. CONCLUSIONS: The antisense PS-ODNs are effective in downregulation of SARS. The findings indicate that antisense knockdown of SARS could be a useful strategy for treatment of SARS, and could also be suitable for studies of the pathological function of SARS genes in a cellular model system.     

Identification of the INO1 gene of Mycobacterium tuberculosis H37Rv reveals a novel class of inositol-1-phosphate synthase enzyme.

1L-myo-inositol (inositol) is vital for the biogenesis of mycothiol, phosphatidylinositol and glycosylphosphatidylinositol anchors linked to complex carbohydrates in Mycobacterium tuberculosis. All these cellular components are thought to play important roles in host-pathogen interactions and in the survival of the pathogen within the host. However, the inositol biosynthetic pathway in M. tuberculosis is not known. To delineate the pathways for inositol formation, we employed a unique combination of tertiary structure prediction and yeast-based functional assays. Here, we describe the identification of the gene for mycobacterial INO1 that encodes inositol-1-phosphate synthase distinct in many respects from the eukaryotic analogues.     

盐地碱蓬(Suaeda salsa)中SsINPS基因的分子克隆及表达特性分析

肌醇 1 磷酸 (I 1 P)合成酶 (EC5 .5 .1 .4,INPS)是肌醇生物合成中的关键酶 ,催化葡萄糖 6 磷酸 (G 6 P)到I 1 P的反应。从该实验室已构建的NaCl40 0mmol/L处理的盐地碱蓬 (Suaedasal sa)cDNA文库中克隆了肌醇 1 磷酸合成酶的全长cDNA (S .salsamyo inositol 1 phosphatesynthase,SsINPS) ,基因注册号为AF43 3 879。SsINPS全长约 1 986bp ,含有开放式阅读框架 1 5 3 0bp ,3′和 5′的非翻译区分别为 1 3 9bp和 3 1 7bp ;推导的氨基酸序列全长 5 1 0个氨基酸残基 ,分子量约为 5 6 .7kD ,pI值为 5 .3 5。BLAST同源性分析表明 ,该cDNA与已报告的冰叶日中花 (Mesembryanthemumcrys tallinum)的INPS基因同源性最高 ,其中 ,核苷酸水平的同源性为 91 % ,氨基酸水平上的同源性为84%。以SsINPS全长cDNA为探针进行的South ern杂交结果表明 ,SsINPS基因在盐地碱蓬基因组中只有一个拷贝 ;Northern结果表明 ,在盐处理(40 0mmol/L的NaCl)下 ,SsINPS在叶中的表达量有显著的增加。从而说明SsINPS在盐胁迫下是上升调节的     

The INO2 and INO4 loci of Saccharomyces cerevisiae are pleiotropic regulatory genes.

We isolated a mutant of Saccharomyces cerevisiae defective in the formation of phosphatidylcholine via methylation of phosphatidylethanolamine. The mutant synthesized phosphatidylcholine at a reduced rate and accumulated increased amounts of methylated phospholipid intermediates. It was also found to be auxotrophic for inositol and allelic to an existing series of ino4 mutants. The ino2 and ino4 mutants, originally isolated on the basis of an inositol requirement, are unable to derepress the cytoplasmic enzyme inositol-1-phosphate synthase (myo-inositol-1-phosphate synthase; EC 5.5.1.4). The INO4 and INO2 genes were, thus, previously identified as regulatory genes whose wild-type product is required for expression of the INO1 gene product inositol-1-phosphate synthase (T. Donahue and S. Henry, J. Biol. Chem. 256:7077-7085, 1981). In addition to the identification of a new ino4-allele, further characterization of the existing series of ino4 and ino2 mutants, reported here, demonstrated that they all have a reduced capacity to convert phosphatidylethanolamine to phosphatidylcholine. The pleiotropic phenotype of the ino2 and ino4 mutants described in this paper suggests that the INO2 and INO4 loci are involved in the regulation of phospholipid methylation in the membrane as well as inositol biosynthesis in the cytoplasm.     

Elimination of 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid 9-phosphate synthase activity from human N-acetylneuraminic acid 9-phosphate synthase by a single mutation

The most commonly occurring sialic acid Neu5Ac (N-acetylneuraminic acid) and its deaminated form, KDN (2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid), participate in many biological functions. The human Neu5Ac-9-P (Neu5Ac 9-phosphate) synthase has the unique ability to catalyse the synthesis of not only Neu5Ac-9-P but also KDN-9-P (KDN 9-phosphate). Both reactions are catalysed by the mechanism of aldol condensation of PEP (phosphoenolpyruvate) with sugar substrates, ManNAc-6-P (N-acetylmannosamine 6-phosphate) or Man-6-P (mannose 6-phosphate). Mouse and putative rat Neu5Ac-9-P synthases, however, do not show KDN-9-P synthase activity, despite sharing high sequence identity (>95%) with the human enzyme. Here, we demonstrate that a single mutation, M42T, in human Neu5Ac-9-P synthase can abolish the KDN-9-P synthase activity completely without compromising the Neu5Ac-9-P synthase activity. Saturation mutagenesis of Met42 of the human Neu5Ac-9-P synthase showed that the substitution with all amino acids except leucine retains only the Neu5Ac-9-P synthase activity at levels comparable with the wild-type enzyme. The M42L mutant, like the wild-type enzyme, showed the additional KDN-9-P synthase activity. In the homology model of human Neu5Ac-9-P synthase, Met42 is located 22 A (1 A=0.1 nm) away from the substrate-binding site and the impact of this distant residue on the enzyme functions is discussed.     

Characterization of a yeast regulatory mutant constitutive for synthesis of inositol-1-phosphate synthase

Summary The enzyme inositol-1-phosphate synthase is repressed at least 50-fold in wild type yeast grown in inositol-supplemented media. Mutants which synthesize this enzyme constitutively have been isolated using a selection procedure based on excretion of inositol into the growth medium by putative mutants. Biochemical analysis of one of the mutants (opi1-1) confirmed that the nature of the mutations is regulatory, and not in the structural gene for the enzyme. Immunoprecipitation of crude extracts with antibody directed against purified inositol-1-phosphate synthase showed that a protein which reacts with the antibody is present in the mutant grown under both repressing and derepressing conditions, in contrast to the wild type which synthesizes the enzyme only when derepressed. Assay of inositol-1-phosphate synthase activity in crude extracts of the mutant verified synthase activity in cells grown under both repressing and drepressing conditions. Synthase purified from this mutant was characterized with respect to molecular weight, thermolability and affinity for substrates glucose-6-phosphate and NAD. These analyses indicated that purified mutant synthase was similar to the wild type enzyme.     

Biosynthesis of Di-myo-Inositol-1,1′-Phosphate,a Novel Osmolyte in Hyperthermophilic Archaea

Biosynthesis of di-myo-inositol-1,1′-phosphate (DIP) is proposed to occur with myo-inositol and myo-inositol 1-phosphate (I-1-P) used as precursors. Activation of the I-1-P with CTP and condensation of the resultant CDP-inositol (CDP-I) with myo-inositol then generates DIP. The sole known biosynthetic pathway of inositol in all organisms is the conversion of d-glucose-6-phosphate to myo-inositol. This conversion requires two key enzymes: l-I-1-P synthase and I-1-P phosphatase. Enzymatic assays using ³¹P nuclear magnetic resonance spectroscopy as well as a colorimetric assay for inorganic phosphate have confirmed the occurrence of l-I-1-P synthase and a moderately specific I-1-P phosphatase. The enzymatic reaction that couples CDP-I with myo-inositol to generate DIP has also been detected in Methanococcus igneus. ¹³C labeling studies with [2,3-¹³C]pyruvate and [3-¹³C]pyruvate were used to examine this pathway in M. igneus. Label distribution in DIP was consistent with inositol units formed from glucose-6-phosphate, but the label in the glucose moiety was scrambled via transketolase and transaldolase activities of the pentose phosphate pathway.Di-myo-inositol-1,1′-phosphate (DIP) is an unusual inositol derivative that has been identified as a major solute in hyperthermophilic archaea including Pyrococcus woesei (22), Pyrococcus furiosus (16), Methanococcus igneus (5), and several eubacteria of the order Thermotogales (15). Intracellular DIP increases with increasing extracellular concentrations of NaCl in both M. igneus (5) and P. furiosus (16). DIP also increases dramatically at supraoptimal growth temperatures (>80°C for M. igneus and 98 to 101°C for P. furiosus). The unusual intracellular high concentration of K⁺ ions and the extreme optimal growth temperatures (100 to 104°C) of P. woesei (30) suggested the role of DIP as a main counterion of K⁺ with a possible thermostabilizing action. Scholz et al. (22) demonstrated that among several salts, the potassium salt of DIP provided optimum enzyme stabilization when the activity of glyceraldehyde-3-phosphate dehydrogenase of P. woesei was tested at 105°C under anaerobic conditions.Since de novo synthesis of DIP occurs in response to external levels of NaCl and temperature, there must be regulatory biosynthetic mechanisms linked to osmotic pressure and temperature. To study the regulation, the enzymes and/or other proteins responsible for synthesis of this compatible solute must be isolated. This requires knowledge of the biosynthetic pathways involved in the synthesis of DIP. The sole known pathway for inositol biosynthesis in all other organisms is the conversion of d-glucose-6-phosphate to l-myo-inositol 1-phosphate (l-I-1-P) via l-myo-inositol 1-monophosphate (I-1-P) synthase and hydrolysis of I-1-P to myo-inositol via a specific phosphatase, I-1-P phosphatase (13, 14). Similar enzymes are likely to exist in methanogens. A logical pathway for the biosynthesis of DIP would then use myo-inositol and I-1-P as precursors. Activation of the I-1-P with CTP and condensation of the resultant CDP-inositol (CDP-I) with myo-inositol would generate DIP. As summarized in Fig. ​Fig.1,1, DIP biosynthesis requires four key enzymes: I-1-P synthase (step 1), I-1-P phosphatase (step 2), CTP:I-1-P cytidylyltransferase (step 3), and DIP synthase (step 4). The enzymes that catalyze steps 1 and 2 have been well studied in plants, yeasts, and mammalian tissues. However, the enzymes invoked for steps 3 and 4 are novel activities, although based on similar chemical transformations in cells. Open in a separate windowFIG. 1Proposed biosynthetic pathway for DIP showing the four key enzymatic activities. Based on similar transformations in other organisms, cofactors are indicated for several of the steps.This work describes the use of ³¹P nuclear magnetic resonance (NMR) and colorimetric assays to verify the existence of three of these activities in cell extracts of M. igneus. Specific labeling of DIP with [¹³C]pyruvate was also used to probe the DIP biosynthetic pathway. The pattern of ¹³C label incorporation from [3-¹³C]pyruvate and [2,3-¹³C]pyruvate coupled with the known stereochemistry of DIP provided evidence that M. igneus also has enzymes of the pentose phosphate pathway (transaldolase and transketolase) that scramble label in glucose-6-phosphate.     

Kinetics of phosphatidylinositol-3-phosphate acquisition differ between IgG bead-containing phagosomes and Mycobacterium tuberculosis-containing phagosomes

A key aspect of Mycobacterium tuberculosis pathogenesis is the ability of the bacteria to survive within the host macrophage. A phagosome containing an IgG-coated bead matures into a lysosomal compartment as evidenced by a decrease in pH and an increased acquisition of hydrolytic enzymes. In contrast, when M. tuberculosis is phagocytosed, the maturation of the bacteria-containing phagosome is arrested, and the bacterium resides within a vacuole that retains characteristics of early endosomal compartments. M. tuberculosis-containing phagosomes are delayed in the recruitment of the early endosome autoantigen EEA1. Acquisition of EEA1 is dependent on the presence of phosphatidylinositol-3-phosphate (PI-3-P) generated by the kinase Vps34. We tested the hypothesis that delayed recruitment of EEA1 was due to altered kinetics of PI-3-P accumulation at the phagosomal membrane. Biochemical analysis of the phosphatidylinositol phosphates on M. tuberculosis-containing phagosomes revealed that PI-3-P acquisition was markedly retarded and reduced in comparison to IgG bead-containing phagosomes. Given the role these lipids play in the regulation of phagosome maturation these findings have implications with respect to the mechanisms behind the arrest of phagosome maturation.     

Effect of growth phase on phospholipid biosynthesis in Saccharomyces cerevisiae.

The effect of growth phase on the membrane-associated phospholipid biosynthetic enzymes CDP-diacylglycerol synthase, phosphatidylserine synthase, phosphatidylinositol synthase, and the phospholipid N-methyltransferases in wild-type Saccharomyces cerevisiae was examined. Maximum activities were found in the exponential phase of cells grown in complete synthetic medium. As cells entered the stationary phase of growth, the activities of the CDP-diacylglycerol synthase, phosphatidylserine synthase, and the phospholipid N-methyltransferases decreased 2.5- to 5-fold. The subunit levels of phosphatidylserine synthase and the cytoplasmic-associated enzyme inositol-1-phosphate synthase were not significantly affected by the growth phase. When grown in medium supplemented with inositol-choline, cells in the exponential phase of growth had reduced CDP-diacylglycerol synthase, phosphatidylserine synthase, and phospholipid N-methyltransferase activities, with repressed subunit levels of phosphatidylserine synthase and inositol-1-phosphate synthase compared with cells grown without inositol-choline. Enzyme activity levels remained reduced in the stationary phase of growth of cells supplemented with inositol-choline. The phosphatidylserine synthase and inositol-1-phosphate synthase subunit levels, however, were depressed. Phosphatidylinositol synthase (activity and subunit) was not affected by growth in medium supplemented with or without inositol-choline or the growth phase of the culture. The phospholipid composition of cells in the exponential and stationary phase of growth was also examined. The phosphatidylinositol to phosphatidylserine ratio doubled in stationary-phase cells. The phosphatidylcholine to phosphatidylethanolamine ratio was not significantly affected by the growth phase of cells.     

Insulin-like growth factor family and combined antisense approach in therapy of lung carcinoma

BACKGROUND: Perturbation in a level of any peptide from insulin-like growth factor (IGF) family (ligands, receptors, and binding proteins) seems to be implicated in lung cancer formation; IGF ligands and IGF-I receptor through their mitogenic and anti-apoptotic action, and the mannose 6-phosphate/insulin-like growth factor II receptor (M6-P/IGF-IIR) possibly as a tumor suppressor. MATERIALS AND METHODS: To determine the identity, role, and mutual relationship of IGFs in lung cancer growth and maintenance, we examined IGF's gene (by RT-PCR) and protein (by immunohistochemistry) expression in 69 human lung carcinoma tissues. We also examined IGF-I receptor numbers (Scatchard analysis) and IGF-II production and release (by Western blot) in IGF-II/IGF-IR mRNA positive and negative lung carcinomas. Finally, the potential role of IGF-IR and IGF-II as growth promoting factors in lung cancer was studied using antisense oligodeoxynucleotides that specifically inhibit IGF-IR and IGF-II mRNA. RESULTS: Thirty-two tumors were positive for IGF-I, 39 for IGF-II, 48 for IGF-IR, and 35 for IGFBP-4 mRNA. Seventeen tumors were concomitantly positive for all four IGFs, whereas 34 were positive for IGF-II, IGF-IR, and IGFBP-4 mRNA. An elevated amount of IGF-II peptide was secreted into the growth medium of cell cultures established from five different IGF-II/IGF-IR mRNA positive lung cancer tissues. The cells also expressed elevated numbers of IGF-IR. Nine IGF-II-negative and 19 IGF-II-positive lung cancers of different stages were selected, and M6-P/ IGF-II receptor was determined immunohistochemically. Most of the IGF-II-negative tumors were strongly positive for M6-P/IGF-IIR. IGF-II-positive tumors were mostly negative for M6-P/IGF-II receptors. Antisense oligodeoxynucleotides to IGF-II significantly inhibited, by 25-60%, the in vitro growth of all six lung cancer cell lines. However, the best results (growth inhibition of up to 80%) were achieved with concomitant antisense treatment (to IGF-IR and IGF-II). CONCLUSION: Our data suggest that lung cancer cells produce IGF-IR and IGF-II, which in turn stimulates their proliferation by autocrine mechanism. Cancer cell proliferation can be abrogated or alleviated by blocking the mRNA activity of these genes indicating that an antisense approach may represent an effective and practical cancer gene therapy strategy.     

Saccharomyces cerevisiae mutant with a partial defect in the synthesis of CDP-diacylglycerol and altered regulation of phospholipid biosynthesis.

A Saccharomyces cerevisiae mutant (cdg1 mutation) was isolated on the basis of an inositol excretion phenotype and exhibited pleiotropic deficiencies in phospholipid biosynthesis. Genetic analysis of the mutant confirmed that the cdg1 mutation represents a new genetic locus and that a defect in a single gene was responsible for the Cdg1 phenotype. CDP-diacylglycerol synthase activity in mutant haploid cells was 25% of the wild-type derepressed level. Biochemical and immunoblot analyses revealed that the defect in CDP-diacylglycerol synthase activity in the cdg1 mutant was due to a reduced level of the CDP-diacylglycerol synthase Mr-56,000 subunit rather than to an alteration in the enzymological properties of the enzyme. This defect resulted in a reduced rate of CDP-diacylglycerol synthesis, an elevated phosphatidate content, and alterations in overall phospholipid synthesis. Unlike wild-type cells, CDP-diacylglycerol synthase was not regulated in response to water-soluble phospholipid precursors. The cdg1 lesion also caused constitutive expression of inositol-1-phosphate synthase and elevated phosphatidylserine synthase. Phosphatidylinositol synthase was not affected in the cdg1 mutant.     

Microarray analysis of the nitrate response in Arabidopsis roots and shoots reveals over 1,000 rapidly responding genes and new linkages to glucose,trehalose-6-phosphate,iron, and sulfate metabolism

The genomic response to low levels of nitrate was studied in Arabidopsis using the Affymetrix ATH1 chip containing more than 22,500 probe sets. Arabidopsis plants were grown hydroponically in sterile liquid culture on ammonium as the sole source of nitrogen for 10 d, then treated with 250 microm nitrate for 20 min. The response to nitrate was much stronger in roots (1,176 genes showing increased or decreased mRNA levels) than in shoots (183 responding genes). In addition to known nitrate-responsive genes (e.g. those encoding nitrate transporters, nitrate reductase, nitrite reductase, ferredoxin reductase, and enzymes in the pentose phosphate pathway), genes encoding novel metabolic and potential regulatory proteins were found. These genes encode enzymes in glycolysis (glucose-6-phosphate isomerase and phosphoglycerate mutase), in trehalose-6-P metabolism (trehalose-6-P synthase and trehalose-6-P phosphatase), in iron transport/metabolism (nicotianamine synthase), and in sulfate uptake/reduction. In many cases, only a few select genes out of several in small gene families were induced by nitrate. These results show that the effect of nitrate on gene expression is substantial (affecting almost 10% of the genes with detectable mRNA levels) yet selective and affects many genes involved in carbon and nutrient metabolism.     

Recombinase-based reporter system and antisense technology to study gene expression and essentiality in hypoxic nonreplicating mycobacteria

The study of the mechanisms used by Mycobacterium tuberculosis to survive in the absence of growth is hampered by the absence of appropriate genetic tools. Here, we report two strategies, a recombinase-based reporter system and an antisense technology, to study gene expression and essentiality in hypoxic nonreplicating mycobacteria. The recombinase-based reporter system relies on the resolution of an antibiotic marker flanked by the gammadelta-res sites. This system was developed to identify M. tuberculosis promoters, which are specifically expressed under anaerobic conditions. The antisense strategy was designed to study the role of a gene candidate during anaerobic survival. To validate this approach, the dosR, narK2 and rv2466c promoters were selected to drive dosR antisense mRNA expression in quiescent mycobacteria. The conditional knockout strains were found to be attenuated to adapt and survive under anaerobic conditions, as observed for the dosR knockout strain. Together, our work demonstrates that the recombinase-based reporter system and antisense technology represent two genetic tools useful for the identification and characterization of genes essential for the survival of hypoxic nonreplicating M. tuberculosis.     

Probing the mechanism of the Archaeoglobus fulgidus inositol-1-phosphate synthase

myo-Inositol-1-phosphate synthase (mIPS) catalyzes the conversion of glucose-6-phosphate (G-6-P) to inositol-1-phosphate. In the sulfate-reducing archaeon Archaeoglobus fulgidus it is a metal-dependent thermozyme that catalyzes the first step in the biosynthetic pathway of the unusual osmolyte di-myo-inositol-1,1'-phosphate. Several site-specific mutants of the archaeal mIPS were prepared and characterized to probe the details of the catalytic mechanism that was suggested by the recently solved crystal structure and by the comparison to the yeast mIPS. Six charged residues in the active site (Asp225, Lys274, Lys278, Lys306, Asp332, and Lys367) and two noncharged residues (Asn255 and Leu257) have been changed to alanine. The charged residues are located at the active site and were proposed to play binding and/or direct catalytic roles, whereas noncharged residues are likely to be involved in proper binding of the substrate. Kinetic studies showed that only N255A retains any measurable activity, whereas two other mutants, K306A and D332A, can carry out the initial oxidation of G-6-P and reduction of NAD+ to NADH. The rest of the mutant enzymes show major changes in binding of G-6-P (monitored by the 31P line width of inorganic phosphate when G-6-P is added in the presence of EDTA) or NAD+ (detected via changes in the protein intrinsic fluorescence). Characterization of these mutants provides new twists on the catalytic mechanism previously proposed for this enzyme.