MJ0109 is an enzyme that is both an inositol monophosphatase and the 'missing' archaeal fructose-1,6-bisphosphatase

In sequenced genomes, protein coding regions with unassigned function constitute between 10 and 50% of all open reading frames. Often key enzymes cannot be identified using sequence homology searches. For example, despite the fact that methanogens have an apparently functional gluconeogenesis pathway, standard tools have been unable to identify a fructose-1,6-bisphosphatase (FBPase) gene in the sequenced Methanoccocus jannaschii genome. Using a combination of functional and structural tools, we have shown that the protein product of the M. jannaschii gene MJ0109, which had been tentatively annotated as an inositol monophosphatase (IMPase), has both IMPase and FBPase activities. Moreover, several gene products annotated as IMPases from different thermophilic organisms also possess FBPase activity. Thus, we have found the FBPase that was 'missing' in thermophiles and shown that it also functions as an IMPase.     

NADP(H) phosphatase activities of archaeal inositol monophosphatase and eubacterial 3'-phosphoadenosine 5'-phosphate phosphatase

NADP(H) phosphatase has not been identified in eubacteria and eukaryotes. In archaea, MJ0917 of hyperthermophilic Methanococcus jannaschii is a fusion protein comprising NAD kinase and an inositol monophosphatase homologue that exhibits high NADP(H) phosphatase activity (S. Kawai, C. Fukuda, T. Mukai, and K. Murata, J. Biol. Chem. 280:39200-39207, 2005). In this study, we showed that the other archaeal inositol monophosphatases, MJ0109 of M. jannaschii and AF2372 of hyperthermophilic Archaeoglobus fulgidus, exhibit NADP(H) phosphatase activity in addition to the already-known inositol monophosphatase and fructose-1,6-bisphosphatase activities. Kinetic values for NADP+ and NADPH of MJ0109 and AF2372 were comparable to those for inositol monophosphate and fructose-1,6-bisphosphate. This implies that the physiological role of the two enzymes is that of an NADP(H) phosphatase. Further, the two enzymes showed inositol polyphosphate 1-phosphatase activity but not 3'-phosphoadenosine 5'-phosphate phosphatase activity. The inositol polyphosphate 1-phosphatase activity of archaeal inositol monophosphatase was considered to be compatible with the similar tertiary structures of inositol monophosphatase, fructose-1,6-bisphosphatase, inositol polyphosphate 1-phosphatase, and 3'-phosphoadenosine 5'-phosphate phosphatase. Based on this fact, we found that 3'-phosphoadenosine 5'-phosphate phosphatase (CysQ) of Escherichia coli exhibited NADP(H) phosphatase and fructose-1,6-bisphosphatase activities, although inositol monophosphatase (SuhB) and fructose-1,6-bisphosphatase (Fbp) of E. coli did not exhibit any NADP(H) phosphatase activity. However, the kinetic values of CysQ and the known phenotype of the cysQ mutant indicated that CysQ functions physiologically as 3'-phosphoadenosine 5'-phosphate phosphatase rather than as NADP(H) phosphatase.     

Unexpected similarity in regulation between an archaeal inositol monophosphatase/fructose bisphosphatase and chloroplast fructose bisphosphatase

Hyperthermophilic archaea have an unusual phosphatase that exhibits activity toward both inositol-1-phosphate and fructose-1,6-bisphosphate, activities carried out by separate gene products in eukaryotes and bacteria. The structures of phosphatases from Archaeoglobus fulgidus (AF2372) and Methanococcus jannaschii (MJ0109), both anaerobic organisms, resemble the dimeric unit of the tetrameric pig kidney fructose bisphosphatase (FBPase). A striking feature of AF2372, but not of MJ0109, is that the sulfhydryl groups of two cysteines, Cys150 and Cys186, are in close proximity (4 A). A similar arrangement of cysteines has been observed in chloroplast FBPases that are regulated by disulfide formation controlled by redox signaling pathways (ferredoxin/thioredoxin). This mode of regulation has not been detected in any other FBPase enzymes. Biochemical assays show that the AF2372 phosphatase activity can be abolished by incubation with O(2). Full activity is restored by incubation with thiol-containing compounds. Neither the C150S variant of AF2372 nor the equivalent phosphatase from M. jannaschii loses activity with oxidation. Oxidation experiments using Escherichia coli thioredoxin, in analogy with the chloroplast FBPase system, indicate an unexpected mode of regulation for AF2372, a key phosphatase in this anaerobic sulfate reducer.     

Crystal structure and catalytic mechanism of the MJ0109 gene product: a bifunctional enzyme with inositol monophosphatase and fructose 1,6-bisphosphatase activities

Inositol monophosphatase (EC 3.1.3.25) in hyperthermophilic archaea is thought to play a role in the biosynthesis of di-myo-inositol-1,1'-phosphate (DIP), an osmolyte unique to hyperthermophiles. The Methanococcus jannaschii MJ109 gene product, the sequence of which is substantially homologous to that of human inositol monophosphatase, exhibits inositol monophosphatase activity but with substrate specificity that is broader than those of bacterial and eukaryotic inositol monophosphatases (it can also act as a fructose bisphosphatase). To understand its substrate specificity as well as the poor inhibition by Li(+) (a potent inhibitor of the mammalian enzyme), we have crystallized the enzyme and determined its three-dimensional structure. The overall fold, as expected, is similar to that of the mammalian enzyme, but the details suggest a closer relationship to fructose 1,6-bisphosphatases. Three complexes of the MJ0109 protein with substrate and/or product and inhibitory as well as activating metal ions suggest that the phosphatase mechanism is a three-metal ion assisted catalysis which is in variance with that proposed previously for the human inositol monophosphatase.     

Rv2131c gene product: an unconventional enzyme that is both inositol monophosphatase and fructose-1,6-bisphosphatase

Inositol monophosphatase is an enzyme in the biosynthesis of myo-inostiol, a crucial substrate for the synthesis of phosphatidylinositol, which has been demonstrated to be an essential component of mycobacteria. In this study, the Rv2131c gene from Mycobacterium tuberculosis H37Rv was cloned into the pET28a vector and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) strain, allowing the expression of the enzyme in fusion with a histidine-rich peptide on the N-terminal. The fusion protein was purified from the soluble fraction of the lysed cells under native conditions by immobilized metal affinity chromatography (IMAC). The purified Rv2131c gene product showed inositol monophosphatase activity but with substrate specificity that was broader than those of several bacterial and eukaryotic inositol monophosphatases, and it also acted as fructose-1,6-bisphosphatase. The dimeric enzyme exhibited dual activities of IMPase and FBPase, with K(m) of 0.22+/-0.03mM for inositol-1-phosphate and K(m) of 0.45+/-0.05mM for fructose-1,6-bisphosphatase. To better understand the relationship between the function and structure of the Rv2131c enzyme, we constructed D40N, L71A, and D94N mutants and purified these corresponding proteins. Mutations of D40N and D94N caused the proteins to almost completely lose both the inositol monophosphatase and fructose-1,6-bisphosphatase activities. However, L71A mutant did not cause loss either of the activities, but the activity toward the inositol was 12-fold more resistant to inhibition by lithium (IC(50) approximately 60mM). Based on the substrate specificity and presence of conserved sequence motifs of the M. tuberculosis Rv2131c, we proposed that the enzyme belonged to class IV fructose-1,6-bisphosphatase (FBPase IV).     

Effect of fructose 2,6-bisphosphate on the kinetic properties of cytoplasmic fructose 1,6-bisphosphatase from germinating castor bean endosperm

The cytoplasmic form of fructose 1,6-bisphosphatase (FBPase) was purified over 60-fold from germinating castor bean endosperm (Ricinus communis). The kinetic properties of the purified enzyme were studied. The preparation was specific for fructose 1,6-bisphosphate and exhibited optimum activity at pH 7.5. The affinity of the enzyme for fructose 1,6-bisphosphate was reduced by AMP, which was a mixed linear inhibitor. Fructose 2,6-bisphosphate also inhibited FBPase and induced a sigmoid response to fructose 1,6-bisphosphate. The effects of fructose 2,6-bisphosphate were enhanced by low levels of AMP. The latter two compounds interacted synergistically in inhibiting FBPase, and their interaction was enhanced by phosphate which, by itself, had little effect. The enzyme was also inhibited by ADP, ATP, UDP and, to a lesser extent, phosphoenolpyruvate. There was no apparent synergism between UDP, a mixed inhibitor, and fructose 2,6-bisphosphate. Similarly ADP, a predominantly competitive inhibitor, did not interact with fructose 2,6-bisphosphate. Possible roles for fructose 2,6-bisphosphate and the other effectors in regulating FBPase are discussed.     

The regulation of the interaction between F-actin and muscle fructose 1,6-bisphosphatase

Interaction between rabbit muscle fructose 1,6-bisphosphatase (FBPase) and rabbit muscle F-actin results in heterologous complex formation [A. Gizak, D. Rakus, A. Dzugaj, Histol. Histopathol. 18 (2003) 135]. Calculated on the basis of co-sedimentation-binding experiments and ELISA assay-binding constant (Ka) revealed that FBPase binds to F-actin with Ka equal to 7.4 x 10(4) M(-1). The binding is down-regulated by ligands interacting with the FBPase active site (fructose 6-phosphate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate) and with the FBPase allosteric inhibitory site (AMP). The binding and the kinetic data suggests that FBPase may bind F-actin using a bipartite motif which includes the amino acids residues involved in the binding of the substrate as well as of the allosteric inhibitor of the enzyme. The in situ co-localization experiment, in which FBPase was diffused into skinned muscle fibres pre-incubated with phalloidin (polymeric actin-interacting toxin), has shown that FBPase binds predominantly to the region of the Z-line.     

Regulatory properties of a fructose 1,6-bisphosphatase from the cyanobacterium Anacystis nidulans.

A fructose 1,6-bisphosphatase (EC 3.1.3.11) (FBPase) was purified over 100-fold from Anacystis nidulans. At variance with a previous report (R. H. Bishop, Arch. Biochem. Biophys. 196:295-300, 1979), the regulatory properties of the enzyme were found to be like those of chloroplast enzymes rather than intermediate between chloroplast (photosynthetic) and heterotrophic FBPases. The pH optimum of Anacystis FBPase was between 8.0 and 8.5 and shifted to lower values with increasing Mg2+ concentration. Under the experimental conditions used by Bishop, we found the saturation curve of the enzyme to be sigmoidal for Mg2+ ions and hyperbolic for fructose 1,6-bisphosphate. The half-maximal velocity of the Anacystis FBPase was reached at concentrations of 5 mM MgCl2 and 0.06 mM fructose 1,6-bisphosphate. AMP did not inhibit the enzyme. The activity of the FBPase was found to be under a delicate control of oxidizing and reducing conditions. Oxidants like O2, H2O2, oxidized glutathione, and dehydroascorbic acid decreased the enzyme activity, whereas reductants like dithiothreitol and reduced glutathione increased it. The oxido-reductive modulation of FBPase proved to be reversible. Reduced glutathione stimulated the enzyme activity at physiological concentrations (1 to 10 mM).l The reduced glutathione-induced activation was higher at pH 8.0 than at pH 7.0.     

NADP(H) Phosphatase Activities of Archaeal Inositol Monophosphatase and Eubacterial 3′-Phosphoadenosine 5′-Phosphate Phosphatase

NADP(H) phosphatase has not been identified in eubacteria and eukaryotes. In archaea, MJ0917 of hyperthermophilic Methanococcus jannaschii is a fusion protein comprising NAD kinase and an inositol monophosphatase homologue that exhibits high NADP(H) phosphatase activity (S. Kawai, C. Fukuda, T. Mukai, and K. Murata, J. Biol. Chem. 280:39200-39207, 2005). In this study, we showed that the other archaeal inositol monophosphatases, MJ0109 of M. jannaschii and AF2372 of hyperthermophilic Archaeoglobus fulgidus, exhibit NADP(H) phosphatase activity in addition to the already-known inositol monophosphatase and fructose-1,6-bisphosphatase activities. Kinetic values for NADP⁺ and NADPH of MJ0109 and AF2372 were comparable to those for inositol monophosphate and fructose-1,6-bisphosphate. This implies that the physiological role of the two enzymes is that of an NADP(H) phosphatase. Further, the two enzymes showed inositol polyphosphate 1-phosphatase activity but not 3′-phosphoadenosine 5′-phosphate phosphatase activity. The inositol polyphosphate 1-phosphatase activity of archaeal inositol monophosphatase was considered to be compatible with the similar tertiary structures of inositol monophosphatase, fructose-1,6-bisphosphatase, inositol polyphosphate 1-phosphatase, and 3′-phosphoadenosine 5′-phosphate phosphatase. Based on this fact, we found that 3′-phosphoadenosine 5′-phosphate phosphatase (CysQ) of Escherichia coli exhibited NADP(H) phosphatase and fructose-1,6-bisphosphatase activities, although inositol monophosphatase (SuhB) and fructose-1,6-bisphosphatase (Fbp) of E. coli did not exhibit any NADP(H) phosphatase activity. However, the kinetic values of CysQ and the known phenotype of the cysQ mutant indicated that CysQ functions physiologically as 3′-phosphoadenosine 5′-phosphate phosphatase rather than as NADP(H) phosphatase.     

Saccharomyces cerevisiae aldolase mutants.

Six mutants lacking the glycolytic enzyme fructose 1,6-bisphosphate aldolase have been isolated in the yeast Saccharomyces cerevisiae by inositol starvation. The mutants grown on gluconeogenic substrates, such as glycerol or alcohol, and show growth inhibition by glucose and related sugars. The mutations are recessive, segregate as one gene in crosses, and fall in a single complementation group. All of the mutants synthesize an antigen cross-reacting to the antibody raised against yeast aldolase. The aldolase activity in various mutant alleles measured as fructose 1,6-bisphosphate cleavage is between 1 to 2% and as condensation of triose phosphates to fructose 1,6-bisphosphate is 2 to 5% that of the wild-type. The mutants accumulate fructose 1,6-bisphosphate from glucose during glycolysis and dihydroxyacetone phosphate during gluconeogenesis. This suggests that the aldolase activity is absent in vivo.     

Characterization of Hyperthermostable Fructose-1,6-Bisphosphatase from <Emphasis Type="Italic">Thermococcus onnurineus</Emphasis> NA1

To understand the physiological functions of thermostable fructose-1,6-bisphosphatase (TNA1-Fbp) from Thermococcus onnurineus NA1, its recombinant enzyme was overexpressed in Escherichia coli, purified, and the enzymatic properties were characterized. The enzyme showed maximal activity for fructose-1,6-bisphosphate at 95°C and pH 8.0 with a half-life (t _1/2) of about 8 h. TNA1-Fbp had broad substrate specificities for fructose-1,6-bisphosphate and its analogues including fructose-1-phosphate, glucose-1-phosphate, and phosphoenolpyruvate. In addition, its enzyme activity was increased five-fold by addition of 1 mM Mg²⁺, while Li⁺ did not enhance enzymatic activity. TNA1-Fbp activity was inhibited by ATP, ADP, and phosphoenolpyruvate, but AMP up to 100 mM did not have any effect. TNA1-Fbp is currently defined as a class V fructose-1,6-bisphosphatase (FBPase) because it is very similar to FBPase of Thermococcus kodakaraensis KOD1 based on sequence homology. However, this enzyme shows a different range of substrate specificities. These results suggest that TNA1-Fbp can establish new criterion for class V FBPases.     

HETEROLOGOUS EXPRESSION OF Escherichia coli FRUCTOSE-1,6-BISPHOSPHATASE IN Corynebacterium glutamicum AND EVALUATING THE EFFECT ON CELL GROWTH AND L-LYSINE PRODUCTION

Fructose-1,6-bisphosphatase (FBPase), which is mainly used to supply NADPH, has an important role in increasing L-lysine production by Corynebacterium glutamicum. However, C. glutamicum FBPase is negatively regulated at the metabolic level. Strains that overexpressed Escherichia coli fructose-1,6-bisphosphatase in C. glutamicum were constructed, and the effects of heterologous FBPase on cell growth and L-lysine production during growth on glucose, fructose, and sucrose were evaluated. The heterologous fructose-1,6-bisphosphatase is insensitive to fructose 1-phosphate and fructose 2,6-bisphosphate, whereas the homologous fructose-1,6-bisphosphatase is inhibited by fructose 1-phosphate and fructose 2,6-bisphosphate. The relative enzyme activity of heterologous fructose-1,6-bisphosphatase is 90.8% and 89.1% during supplement with 3 mM fructose 1-phosphate and fructose 2,6-bisphosphate, respectively. Phosphoenolpyruvate is an activator of heterologous fructose-1,6-bisphosphatase, whereas the homologous fructose-1,6-bisphosphatase is very sensitive to phosphoenolpyruvate. Overexpression of the heterologous fbp in wild-type C. glutamicum has no effect on L-lysine production, but fructose-1,6-bisphosphatase activities are increased 9- to 13-fold. Overexpression of the heterologous fructose-1,6-bisphosphatase increases L-lysine production in C. glutamicum lysC ^T311I by 57.3% on fructose, 48.7% on sucrose, and 43% on glucose. The dry cell weight (DCW) and maximal specific growth rate (μ) are increased by overexpression of heterologous fbp. A “funnel-cask” diagram is first proposed to explain the synergy between precursors supply and NADPH supply. These results lay a definite theoretical foundation for breeding high L-lysine producers via molecular target.     

Crystal structure of a dual activity IMPase/FBPase (AF2372) from Archaeoglobus fulgidus. The story of a mobile loop

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.     

Kinetic analysis of PPi-dependent phosphofructokinase from Porphyromonas gingivalis

We have previously cloned the gene encoding a pyrophosphate-dependent phosphofructokinase (PFK), designated PgPFK, from Porphyromonas gingivalis, an oral anaerobic bacterium implicated in advanced periodontal disease. In this study, recombinant PgPFK was purified to homogeneity, and biochemically characterized. The apparent K(m) value for fructose 6-phosphate was 2.2 mM, which was approximately 20 times higher than that for fructose 1,6-bisphosphate. The value was significantly greater than any other described PFKs, except for Amycolatopsis methanolica PFK which is proposed to function as a fructose 1,6 bisphosphatase (FBPase). The PgPFK appears to serves as FBPase in this organism. We postulate that this may lead to the gluconeogenic pathways to synthesize the lipopolysaccharides and/or glycoconjugates essential for cell viability.     

Novel allosteric activation site in Escherichia coli fructose-1,6-bisphosphatase

Fructose-1,6-bisphosphatase (FBPase) governs a key step in gluconeogenesis, the conversion of fructose 1,6-bisphosphate into fructose 6-phosphate. In mammals, the enzyme is subject to metabolic regulation, but regulatory mechanisms of bacterial FBPases are not well understood. Presented here is the crystal structure (resolution, 1.45A) of recombinant FBPase from Escherichia coli, the first structure of a prokaryotic Type I FBPase. The E. coli enzyme is a homotetramer, but in a quaternary state between the canonical R- and T-states of porcine FBPase. Phe(15) and residues at the C-terminal side of the first alpha-helix (helix H1) occupy the AMP binding pocket. Residues at the N-terminal side of helix H1 hydrogen bond with sulfate ions buried at a subunit interface, which in porcine FBPase undergoes significant conformational change in response to allosteric effectors. Phosphoenolpyruvate and sulfate activate E. coli FBPase by at least 300%. Key residues that bind sulfate anions are conserved among many heterotrophic bacteria, but are absent in FBPases of organisms that employ fructose 2,6-bisphosphate as a regulator. These observations suggest a new mechanism of regulation in the FBPase enzyme family: anionic ligands, most likely phosphoenolpyruvate, bind to allosteric activator sites, which in turn stabilize a tetramer and a polypeptide fold that obstructs AMP binding.     

L-Lactate dehydrogenase from Thermus caldophilus GK24, an extremely thermophilic bacterium. Desensitization to fructose 1,6-bisphosphate in the activated state by arginine-specific chemical modification and the N-terminal amino acid sequence

Heat-stable and fructose-1,6-bisphosphate-activated L-lactate dehydrogenase (EC 1.1.1.27) has been purified from an extremely thermophilic bacterium, Thermus caldophilus GK24 [Taguchi, H., Yamashita, M., Matsuzawa, H. and Ohta, T. (1982) J. Biochem. (Tokyo) 91, 1343-1348]. N-terminal sequence analysis of the first 34 amino acids of the enzyme indicates that the N-terminal arm region (first 1-20 residues) known for the vertebrate L-lactate dehydrogenases is completely missing in the T. caldophilus enzyme, while there is a high homology of sequence between the regions which are considered to be part of the NAD-binding domain. The C-terminal amino acid of the enzyme was phenylalanine. Analysis of the amino acid composition showed that T. caldophilus enzyme contained much more arginine and fewer lysine than other bacterial and vertebrate L-lactate dehydrogenases. On modification reaction with 2,3-butanedione in the presence of NADH and oxamate, an enhanced activity of the T. caldophilus L-lactate dehydrogenase was obtained independently of fructose 1,6-bisphosphate, and the modified enzyme was desensitized to fructose 1,6-bisphosphate. Amino acid analysis indicated that such a desensitization in the active state was caused by the modification of only one arginine residue per the enzyme subunit. Desensitization of the enzyme was inhibited in the presence of fructose 1,6-bisphosphate. A similar desensitization was observed using 1,2-cyclohexanedione instead of 2,3-butanedione. The enzyme was irreversibly modified with 2,3-butanedione and characterized. The irreversibly modified enzyme also showed an enhanced activity independently of fructose 1,6-bisphosphate, and its pyruvate saturation curve was similar to that of the native enzyme measured in the presence of fructose 1,6-bisphosphate. Fructose 1,6-bisphosphate, which increases the thermostability of the native enzyme, did not affect that of the modified enzyme, while thermostability of the modified enzyme slightly decreased. Amino acid analysis indicated that only the arginine content was decreased by the modification. These results show that arginine residue(s) exist in the binding site for fructose 1,6-bisphosphate on the enzyme, and that the arginine residue(s) play some important role in the allosteric regulation of the enzyme activity.     

The inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate is enhanced by EDTA and diminished by zinc(II)

The sensitivity of the Mg(II)-dependent activity of rabbit liver fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) to inhibition by fructose 2,6-bisphosphate (Fru-2,6-P2) was enhanced by EDTA and diminished to negligible levels by 0.5-2 microM Zn(II) added as another FBPase inhibitor. Fru-2,6-P2 was more efficient in the presence of the synergistic effector AMP: still, the Fru-2,6-P2 concentration inhibiting 50% changed from 3 microM (with EDTA) to higher than 50 microM (with Zn(II]. On the other hand, the Zn(II)-dependent FBPase activity was inhibited by Fru-2,6-P2 to a much lesser extent than the Mg(II)-dependent activity.     

Antagonistic effects of hypertrehalosemic neuropeptide on the activities of 6-phosphofructo-1-kinase and fructose-1,6-bisphosphatase in cockroach fat body

Hypertrehalosemic neuropeptides from the corpora cardiaca such as the decapeptide Bld HrTH bring about a profound switch in the metabolic activity of cockroach fat body during which production of the blood sugar trehalose is stimulated while the catabolism of carbohydrate (glycolysis) is inhibited. The mechanisms of the metabolic switch are not fully understood.Incubation of isolated fat body from the cockroach Blaptica dubia with 10(-8) M Bld HrTH, for 10-60 min, stimulated glycogen breakdown and increased the content of the substrates of both the glycolytic enzyme 6-phosphofructo-1-kinase (PFK, EC 2.7.1.11) and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase, EC 3.1.3.11) in the tissue. The glycolytic signal fructose 2,6-bisphosphate was markedly decreased in fat body on incubation with Bld HrTH. The content of ATP was slightly reduced, while the contents of ADP and AMP were increased after incubation with the hormone.Fructose 2,6-bisphosphate is a potent activator of PFK and a strong inhibitor of FBPase purified from fat body. The activity of PFK was decreased by about 90% when the hormone-dependent changes in effectors and substrates in fat body were simulated in vitro. FBPase, in contrast, was activated about 25-fold under these conditions, suggesting the hormone to stimulate gluconeogenesis in fat body. The data support the view that fructose 2,6-bisphosphate is a pivotal intracellular messenger in the hormone-induced metabolic switch from carbohydrate degradation to trehalose production in cockroach fat body.