Phosphomannose Isomerase Research Articles

A non-antibiotic selection system uses the phosphomannose-isomerase (PMI) gene forAgrobacterium-mediated transformation of Chinese cabbage

To establish a non-antibiotic selection system that utilizes the phosphomannose-isomerase (PMI) gene for Chinese cabbage transformation, we first determined the optimum mannose concentration for selecting transformed cells. Hypocotyl and cotyledon expiants that were grown on media containing more than 5 g L-1 mannose did not induce green calli but, rather became chlorotic and withered before dying. In contrast, media containing 20 g L-1 sucrose plus 5 g L-1 mannose proved suitable for selection. We then used this particular level of mannose to transform hypocotyl tissues. Within 6 weeks, shoots were regenerated from some of the calli; subsequently, these plants were transplanted to pots and grown in the greenhouse. A 514-bp PCR fragment was obtained from most transformants but not from the non-transformed plants. Southern blot analysis also revealed the expectedPMI gene in those PCR-confirmed transgenic plants. RT-PCR of total RNA was performed to confirmPMI expression. We have now demonstrated that this gene does not inhibit the growth of transgenic plants, and that this selection system can be applied to Chinese cabbage transformation.

Medicinal Chemistry of Ureido Derivatives as Anti-Infectives

Ureides are compounds, which essentially incorporate urea as a substructural component either in open or cyclic form. Ureido derivatives are one of the oldest classes of bioactive compounds, widely used as anti-infective agents. Several of these compounds, including aminoquinuride, aminocarbalide, imidurea, cloflucarban, nitrofurazone, urosulfan, are viomycin are used in clinical situations. One of the ureides, the triclocarban is compulsorily used as antibacterial agent in cleansing and disinfecting solutions in hospitals, household, cosmetics, toys, textiles and plastics. It disables the activity of ENR, an enzyme vital for building the cell wall of the bacteria and fungus. Besides, the ureido-penicillins in clinical use, there have been several ureido-lactam derivatives which have been reported to exhibit significant antibacterial activity. A urea containing dipeptide TAN-1057A isolated from Flexibacter spp. has potent bioactivity against MRSA. The metal complexes of sulfonyl ureido derivatives are effective antifungal agents by inhibiting the activity of phosphomannose isomerase, a key enzyme in the biosynthesis of yeast cell walls. There have been a number of ureides including the cyclic ureas, which are potent HIV protease inhibitors and display significant anti-HIV activity. The urea derivative, merimepodib that has been derived using structure based design, is a potent inhibitor of IMPDH and is active against hepatitis-C infection. This review will primarily focus on the significant work reported for this class of compounds including design, synthesis and biological activity. Keywords: antibacterial activity, Enterococcus, topoisomerase, Heterocyclic antibacterial ureides, penicillins

Ablation of Mouse Phosphomannose Isomerase (Mpi) Causes Mannose 6-Phosphate Accumulation, Toxicity, and Embryonic Lethality

MPI encodes phosphomannose isomerase, which interconverts fructose 6-phosphate and mannose 6-phosphate (Man-6-P), used for glycoconjugate biosynthesis. MPI mutations in humans impair protein glycosylation causing congenital disorder of glycosylation Ib (CDG-Ib), but oral mannose supplements normalize glycosylation. To establish a mannose-responsive mouse model for CDG-Ib, we ablated Mpi and provided dams with mannose to rescue the anticipated defective glycosylation. Surprisingly, although glycosylation was normal, Mpi(-/-) embryos died around E11.5. Mannose supplementation even hastened their death, suggesting that man-nose was toxic. Mpi(-/-) embryos showed growth retardation and placental hyperplasia. More than 90% of Mpi(-/-) embryos failed to form yolk sac vasculature, and 35% failed chorioallantoic fusion. We generated primary embryonic fibroblasts to investigate the mechanisms leading to embryonic lethality and found that mannose caused a concentration- and time-dependent accumulation of Man 6-P in Mpi(-/-) fibroblasts. In parallel, ATP decreased by more than 70% after 24 h compared with Mpi(+/+) controls. In cell lysates, Man-6-P inhibited hexokinase (70%), phosphoglucose isomerase (65%), and glucose-6-phosphate dehydrogenase (85%), but not phosphofructokinase. Incubating intact Mpi(-/-) fibroblasts with 2-[(3)H]deoxyglucose confirmed mannose-dependent hexokinase inhibition. Our results in vitro suggest that mannose toxicity in Mpi(-/-) embryos is caused by Man-6-P accumulation, which inhibits glucose metabolism and depletes intracellular ATP. This was confirmed in E10.5 Mpi(-/-) embryos where Man-6-P increased more than 10 times, and ATP decreased by 50% compared with Mpi(+/+) littermates. Because Mpi ablation is embryonic lethal, a murine CDG-Ib model will require hypomorphic Mpi alleles.

Computational study of human phosphomannose isomerase: Insights from homology modeling and molecular dynamics simulation of enzyme bound substrate

Phosphomannose isomerase is a zinc metalloenzyme that catalyzes the reversible isomerization of mannose-6-phosphate and fructose-6-phosphate, and the three-dimensional (3D) structure of human phosphomannose isomerase has not been reported. In order to understand the catalytic mechanism, the 3D structure of the protein is built by using homology modeling based on the known crystal structure of mannose-6-phosphate isomerase from (PDB code 1PMI). The model structure is further refined by energy minimization and molecular dynamics methods. The mannose-6-phosphate–enzyme complex is developed by molecular docking and the key residues involved in the ligand binding are determined, which will facilitate the understanding of the action mode of the ligands and guide further genetic studies. Our results suggest a hydride transfer mechanism of α-hydrogen between the C1 and C2 positions but do not support the cis-enediol mechanism. The detailed mechanism involves, on one side, Zn 2+ mediating the movement of a proton between O1 and O2, and, on the other side, the hydrophobic environment formed in part by Tyr 278 promoting transfer of a hydride ion.

Development of a Genetic Assay to Distinguish between Leishmania viannia Species on the Basis of Isoenzyme Differences

Tegumentary leishmaniasis in Latin America is caused mainly by Leishmania viannia braziliensis complex parasites. L. braziliensis and Leishmania viannia peruviana are the 2 predominant Leishmania species in Peru. L. braziliensis is more virulent, because it can cause mucocutaneous leishmaniasis, known as espundia, that results in severe facial destruction. Early identification of the species that causes the initial cutaneous infection would greatly help to prevent mucocutaneous leishmaniasis, because it would allow more aggressive treatment and follow-up. However, because of the close genetic similarity of L. braziliensis and L. peruviana, there currently exists no simple assay to distinguish between these species. We cloned the mannose phosphate isomerase gene from both L. braziliensis and L. peruviana. It is the only known isoenzyme capable of differentiating between L. braziliensis and L. peruviana in multilocus enzyme electrophoresis. Interestingly, only a single nucleotide polymorphism was found between the mannose phosphate isomerase genes from L. braziliensis and L. peruviana, resulting in an amino acid change from threonine to arginine at amino acid 361. A polymerase chain reaction assay was developed to distinguish the single nucleotide polymorphism of the mannose phosphate isomerase gene to allow for the specific identification of L. braziliensis or L. peruviana. This assay was validated with 31 reference strains that were previously typed by multilocus enzyme electrophoresis, successfully applied to patient biopsy samples, and adapted to a real-time polymerase chain reaction assay. This innovative approach combines new genetic knowledge with traditional biochemical fundamentals of multilocus enzyme electrophoresis to better manage leishmaniasis in Latin America.

Cloning of the aceF gene encoding the phosphomannose isomerase and GDP-mannose pyrophosphorylase activities involved in acetan biosynthesis in Acetobacter xylinum.

The aceF gene from Acetobacter xylinum was identified and cloned from a genomic DNA library. The complete DNA sequence was determined and computer analysis of the translated gene sequence revealed homology with the deduced amino acid sequence of xanB from Xanthomonas campestris. Therefore aceF is likely to encode a bifunctional enzyme with mannose-6-phosphate isomerase (PMI) and GDP-mannose pyrophosphorylase (GMP) activities. PMI and GMP activities were detected in strains of Escherichia coli expressing the cloned aceF gene.

Preliminary studies on the inhibition of D-sorbitol-6-phosphate 2–dehydrogenase from Escherichia coli with substrate analogues

D-Sorbitol-6-phosphate 2-dehydrogenase catalyzes the NADH-dependent conversion of D-fructose 6-phosphate to D-sorbitol 6-phosphate and improved production and purification of the enzyme from Escherichia coli is reported. Preliminary inhibition studies of the enzyme revealed 5-phospho-D-arabinonohydroxamic acid and 5-phospho-D-arabinonate as new substrate analogue inhibitors of the F6P catalyzed reduction with IC50 values of (40 ± 1) μM and (48 ± 3) μM and corresponding Km/IC50 ratio values of 14 and 12, respectively. Furthermore, we report here the phosphomannose isomerase substrate D-mannose 6-phosphate as the best inhibitor of E. coli D-sorbitol-6-phosphate 2-dehydrogenase yet reported with an IC50 = 7.5 ± 0.4 μM and corresponding Km/IC50 ratio = about 76.

Agrobacterium and biolistic transformation of onion using non-antibiotic selection marker phosphomannose isomerase

A new selection system for onion transformation that does not require the use of antibiotics or herbicides was developed. The selection system used the Escherichia coli gene that encodes phosphomannose isomerase (pmi). Transgenic plants carrying the manA gene that codes for pmi can detoxify mannose-6-phosphate by conversion to fructose-6-phosphate, an intermediate of glycolysis, via the pmi activity. Six-week-old embryogenic callus initiated from seedling radicle was used for transformation. Transgenic plants were produced efficiently with transformation rates of 27 and 23% using Agrobacterium and biolistic system, respectively. Untransformed shoots were eliminated by a stepwise increase from 10 g l(-1) sucrose with 10 g l(-1) mannose in the first selection to only 10 g l(-1) mannose in the second selection. Integrative transformation was confirmed by PCR, RT-PCR and Southern hybridization.

Phosphomannose isomerase, pmi, as a selectable marker gene for durum wheat transformation

Transgene technology provides a powerful tool for developing traits that are otherwise difficult to achieve through conventional methods. The development of transgenic plants requires the use of selectable marker genes, as the efficiency of plant transformation is less than optimal for many important species, especially for monocots such as durum wheat ( Triticum turgidum var. durum). Many concerns have been expressed about the persistence of currently used marker genes in plants used for field cultivation. To sustain further progress in this area, alternative efficient selection methods are desirable. A recent development is the use of selective genes that give transformed cells a metabolic advantage (positive selection) compared to untransformed cells, which are slowly starved with a concomitant reduction in growth and viability. This selection strategy is in contrast to traditional negative selections during which the transgenic cells are able to survive on a selective medium whereas the non-transgenic cells are actively killed by the selective agent. We compared the ‘selection efficiency’ of a commonly used negative selection method that employs the Streptomyces hygroscopicus bar gene to confer resistance to the herbicide bialaphos, to a positive selection employing the Escherichia coli phosphomannose isomerase ( pmi) gene as the selectable gene and mannose as the selective agent. Calli derived from immature embryos of the durum wheat cultivar Svevo were bombarded separately with bar and pmi genes using a biolistic system. The integration and expression of the two genes in the T 0 generation were confirmed by PCR analysis with specific primers for each gene and the chlorophenol red assay, respectively. The selection efficiency, calculated as the number of expressing plants divided by the number of total regenerants, was higher when pmi was used as the selectable marker gene (90.1%) than when bar was used (26.4%). Thus, an efficient selection method for durum wheat transformation was established that obviates the use of herbicide résistance genes.

Agrobacterium tumefaciens‐mediated sorghum transformation using a mannose selection system

A dual-marker plasmid containing the selectable marker gene, manA, and the reporter gene, sgfp, was used to transform immature sorghum embryos by employing an Agrobacterium-mediated system. Both genes were under the control of the ubi1 promoter in a binary vector pPZP201. The Escherichia coli phosphomannose isomerase (PMI) gene, pmi, was used as the selectable marker gene and mannose was used as the selective agent. The sgfp gene encoding green fluorescence protein (GFP) was the reporter gene and served as a visual screening marker. A total of 167 transgenic plants were obtained from nine different embryogenic callus lines grown on a selection medium containing 1%-2% mannose. Embryoids and shoots regenerated via embryogenesis, that showed strong GFP fluorescence, were selected from two sorghum genotypes: C401, an inbred line, and Pioneer 8505, a commercial hybrid. The GFP accumulation in transgenic plants was observed with a dissecting stereomicroscope. The integration and expression of the manA gene was confirmed by Southern blot and Western blot analyses, and the feasibility of manA selection was demonstrated by the chlorophenol red (CPR) assay. Our results indicated that transgenes segregated in the Mendelian fashion in the T1 generation. The conversion of mannose to a metabolizable fructose carbon source is beneficial to plants. In addition, except in soybean and a few legumes, no endogenous PMI activity has been detected in plant species, indicating that PMI is useful in the transformation of sorghum. In addition, PMI has no sequence homology to known allergens. Optimization of this selection system for sorghum transformation provides an efficient way to produce transgenic plants without using antibiotic or herbicidal agents as selectable markers, and our results showed that the transformation efficiency reached 2.88% for Pioneer 8505 and 3.30% for C401, both values higher than in previously published reports.

In vivo performance of a dual genetic marker,manA-gfp, in transgenic bentgrass

A dual-marker combination, manA-gfp, comprising 2 independent expression cassettes of genes encoding an Escherichia coli phosphomannose isomerase (PMI) and a synthetic green fluorescent protein (GFP), was incorporated into the binary vector pPZP201. Agrobacterium tumefaciens-mediated transfer was used to introduce the manA-gfp into the mature-seed derived calli of Agrostis stoloifera L. 'Crenshaw'. The putative transgenic bentgrass calli were screened in Murashige and Skoog medium containing 15 g mannose/L, in conjunction with a visual examination of the GFP expression with a fluorescence stereomicroscope. Calli with GFP fluorescence grew well on the mannose selection media. A total of 24 transgenic plants derived from a single piece of callus lobe were studied for the genomic integration, expression, and function of the transgene. Genomic integration of the dual markers manA and gfp was confirmed by Southern blotting analysis, and the expression of manA also was validated by using PMI-specific antiserum. The inheritance and expression of the dual marker, manA-gfp, was demonstrated in the T1 generation. This study on the environmentally friendly markers further documented the feasibility of using alternative selection methods without using herbicide- or antibiotic-resistance markers.

Mechanism and Kinetics of Metalloenzyme Phosphomannose Isomerase: Measurement of Dissociation Constants and Effect of Zinc Binding Using ESI-FTICR Mass Spectrometry

Electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry was used to study the noncovalent complexation of a metalloenzyme, phosphomannose isomerase (PMI), which catalyzes the interconversion of mannose 6-phosphate and fructose 6-phosphate. The zinc cofactor binding effect and the noncovalent interactions of the holoenzyme with its two natural substrates and two inhibitors, erythrose 4-phosphate and mannitol 1-phosphate, were investigated. Under nondenaturing conditions, the intact zinc-containing monomeric protein ions were reproducibly observed with no dissociation. Molecular ions corresponding to apo-PMI monomer were obtained by dialyzing the holoenzyme against EDTA. The binding/release of the metal ion did not alter the charge-state distributions of the protein to any significant extent, but changed the binding affinity of the substrates by at least 5-fold. Using ESI-FTICR mass spectrometry, the binding stoichiometry and specificity of the enzyme-substrate and enzyme-inhibitor complexes were directly determined. The first time report of the apparent dissociation constant for the isomeric substrates of PMI was measured to be 88.8 microM. The relative dissociation constant of the two inhibitors derived from gas-phase noncovalent complexation was very similar to the relative inhibition constant derived from solution phase kinetics.

Isolation by distance and a chromosomal cline in the Cayapa cytospecies of Simulium exiguum, the vector of human onchocerciasis in Ecuador

The population genetic structure of the Cayapa cytospecies of Simulium exiguum, the vector of onchocerciasis, was analysed using allozyme frequency and chromosomal inversion polymorphism data from 6 and 15 populations respectively, collected in Ecuador. Eight allozyme loci were scored. No unique allozyme markers were found enabling us to identify biting adults of the vector from the non-vector Bucay cytotype. Mannose-phosphate isomerase (Mpi) contributed largely to the significant heterogeneity in gene frequency among populations of the Cayapa cytospecies and also to the overall population structuring (F(ST) = 0.015 +/- 0.014) which fitted the isolation by distance model. However, heterozygote deficits were recorded for Mpi in four of the six populations, which could indicate that selection is acting at this locus but this hypothesis will require further convincing evidence. Furthermore the significant population structuring of allozymes was not evident when Mpi was omitted from the analysis. All inversion polymorphisms (IIS-B, IIS-F, IIL-A and IIL-B) were in Hardy-Weinberg equilibrium, showed significant heterogeneity between populations and revealed the occurrence of an altitudinal cline in inversion IIS-B frequency. The inversion polymorphisms revealed a significant degree of population structuring (F(ST) = 0.083 +/- 0.027), which can be explained by the isolation by distance model. A UPGMA cluster analysis revealed the relatively remote, high altitude Rio Mira populations to be the most genetically distinct.

Effective selection of transgenic papaya plants with the PMI/Man selection system

The selectable marker gene phospho-mannose isomerase (pmi), which encodes the enzyme phospho-mannose isomerase (PMI) to enable selection of transformed cell lines on media containing mannose (Man), was evaluated for genetic transformation of papaya (Carica papaya L.). We found that papaya embryogenic calli have little or no PMI activity and cannot utilize Man as a carbon source; however, when calli were transformed with a pmi gene, the PMI activity was greatly increased and they could utilize Man as efficiently as sucrose. Plants regenerated from selected callus lines also exhibited PMI activity but at a lower specific activity level. Our transformation efficiency with Man selection was higher than that reported using antibiotic selection or with a visual marker. For papaya, the PMI/Man selection system for producing transgenic plants is a highly efficient addition to previously published methods for selection and may facilitate the stacking of multiple transgenes of interest. Additionally, since the PMI/Man selection system does not involve antibiotic or herbicide resistance genes, its use might reduce environmental concerns about the potential flow of those genes into related plant populations.

Positive selection.

The use of a new mode of selection-positive selection-has been demonstrated to be successful in a large variety of monocot and dicot species. This selection differs from more traditional modes of selection in which compounds such as antibiotics or herbicides are used to kill nontransformed cells (negative selection). In the case of positive selection, a transformed cell acquires the ability to metabolize a substrate that it previously could not use (or not use efficiently) and thereby grows out of the mass of nontransformed tissue. Positive selection can be of many types from inactive forms of plant growth regulators that are then converted to active forms by the transferred enzyme to alternative carbohydrate sources that are not utilized efficiently by the nontransformed cells that become available upon transformation with an enzyme that allows them to be metabolized. Nontransformed cells either grow slowly in comparison to transformed cells or not at all. Using positive selection, nontransformed cells may die, but, typically, production of phenolic compounds observed with negative selection markers does not occur. In many cases, this effect contributes to higher transformation efficiencies, as these compounds can negatively influence the growth of transformed cells. The use of one form of positive selection-transformation with phosphomannose isomerase followed by selection on mannose containing media-is presented here as an example.

Excess Mannose Limits the Growth of Phosphomannose Isomerase PMI40 Deletion Strain of Saccharomyces cerevisiae

Phosphomannose isomerase (PMI40) catalyzes the conversion between fructose 6-phosphate and mannose 6-phosphate and thus connects glycolysis, i.e. energy production and GDP-mannose biosynthesis or cell wall synthesis in Saccharomyces cerevisiae. After PMI40 deletion (pmi(-)) the cells were viable only if fed with extracellular mannose and glucose. In an attempt to force the GDP-mannose synthesis in the pmi(-) strain by increasing the extracellular mannose concentrations, the cells showed significantly reduced growth rates without any alterations in the intracellular GDP-mannose levels. To reveal the mechanisms resulting in reduced growth rates, we measured genome-wide gene expression levels, several metabolite concentrations, and selected in vitro enzyme activities in central metabolic pathways. The increasing of the initial mannose concentration led to an increase in the mannose 6-phosphate concentration, which inhibited the activity of the second enzyme in glycolysis, i.e. phosphoglucose isomerase converting glucose 6-phosphate to fructose 6-phosphate. As a result of this limitation, the flux through glycolysis was decreased as was the median expression of the genes involved in glycolysis. The expression levels of RAP1, a transcription factor involved in the regulation of the mRNA levels of several enzymes in glycolysis, as well as those of cell cycle regulators CDC28 and CLN3, decreased concomitantly with the growth rates and expression of many genes encoding for enzymes in glycolysis.

Kinetic measurements of phosphoglucose isomerase and phosphomannose isomerase by direct analysis of phosphorylated aldose–ketose isomers using tandem mass spectrometry

A mass spectrometry based method for the direct determination of kinetic constants for phosphoglucose isomerase (PGI) and phosphomannose isomerase (PMI) is described. PGI catalyzes the interconversion between glucose-6-phosphate (Glc6P) and fructose-6-phosphate (Fru6P) and PMI performs the same function between mannose-6-phosphate (Man6P) and Fru6P. These two enzymes are essential in the pathways of glycolytic or oxidative metabolism of carbohydrates and have been considered as potential therapeutic targets. Traditionally, they are assayed either by spectrophotometric detection of Glc6P with one or more coupling enzymes or by a colorimetric detection of Fru6P. However, no suitable assay for Man6P has been developed yet to study the reaction of PMI in the direction from Fru6P to Man6P. In the work presented herein, a general assay for the isomeric substrate-product pair between Glc6P and Fru6P or between Man6P and Fru6P was developed, with the aim of directly studying the kinetics of PGI and PMI in both directions. The 6-phosphorylated aldose and ketose isomers were distinguished based on their corresponding tandem mass spectra (MS 2) obtained on a quadrupole ion trap mass spectrometer, and a multicomponent quantification method was utilized to determine the composition of binary mixtures. Using this method, the conversion between Fru6P and Glc6P and that between Fru6P and Man6P are directly monitored. The equilibrium constants for the reversible reactions catalyzed by PGI and PMI are measured to be 0.3 and 1.1, respectively, and the kinetic parameters for both substrates of PGI and PMI are also determined. The values of K M and V max for Fru6P as substrate of PMI are reported to be 0.15 mM and 7.78 μmol/(min mg), respectively. All other kinetic parameters measured correlate well with those obtained using traditional methods, demonstrating the accuracy and reliability of this assay.

Structural Basis for Phosphomannose Isomerase Activity in Phosphoglucose Isomerase from Pyrobaculum aerophilum: A Subtle Difference between Distantly Related Enzymes

The crystal structure of a dual-specificity phosphoglucose/phosphomannose isomerase from the crenarchaeon Pyrobaculum aerophilum (PaPGI/PMI) has been determined in complex with glucose 6-phosphate at 1.16 A resolution and with fructose 6-phosphate at 1.5 A resolution. Subsequent modeling of mannose 6-phosphate (M6P) into the active site of the enzyme shows that the PMI activity of this enzyme may be due to the additional space imparted by a threonine. In PGIs from bacterial and eukaryotic sources, which cannot use M6P as a substrate, the equivalent residue is a glutamine. The increased space may permit rotation of the C2-C3 bond in M6P to facilitate abstraction of a proton from C2 by Glu203 and, after a further C2-C3 rotation of the resulting cis-enediolate, re-donation of a proton to C1 by the same residue. A proline residue (in place of a glycine in PGI) may also promote PMI activity by positioning the C1-O1 region of M6P. Thus, the PMI reaction in PaPGI/PMI probably uses a cis-enediol mechanism of catalysis, and this activity appears to arise from a subtle difference in the architecture of the enzyme, compared to bacterial and eukaryotic PGIs.

Protective effects of allozyme genotype during chemical exposure in the grass shrimp, Palaemonetes pugio

The primary objective of this research was to determine if the genotype of the enzymes glucose phosphate isomerase ( Gpi), phosphoglucomutase ( Pgm), or mannose phosphate isomerase ( Mpi) are factors affecting survival during acute endosulfan, fluoranthene, and chromium(VI) exposures. Palaemonetes pugio were exposed in the laboratory to 6.3 μg/L endosulfan, 100 mg/L chromium(VI), or 0.6 mg/L fluoranthene. Dead shrimp were removed at approximately 15–30 min intervals and the individual's genotypes for the Gpi, Mpi, and Pgm enzymes were determined. These data were used to establish whether allozyme genotype was related to tolerance as analyzed with non-parametric methods (i.e. development of survival curves using the Kaplan–Meier method of estimation) and parametric methods (i.e. proportional hazards models). Since the sex and/or size of the organism was expected to affect tolerance, these variables were accounted for in the proportional hazards models. Results indicated individuals that were heterozygous (MF) for the Gpi allozyme survived longer and had less overall mortality than the homozygous MM genotype when exposed to chromium(VI) and to fluoranthene. No allozyme genotypes were related to tolerance during the endosulfan exposure when sex was included as a covariate in the analysis. These results support the hypothesis that there is a genetic basis for tolerance in P. pugio during acute exposures to chromium(VI) and to fluoranthene. Although a relationship between Gpi genotype and contaminant tolerance has been identified in previous studies, no such relationship has been documented in P. pugio or with chromium(VI) as the contaminant.

A Novel Phosphoglucose Isomerase (PGI)/Phosphomannose Isomerase from the Crenarchaeon Pyrobaculum aerophilum Is a Member of the PGI Superfamily: STRUCTURAL EVIDENCE AT 1.16-Å RESOLUTION

The crystal structure of a dual specificity phosphoglucose isomerase (PGI)/phosphomannose isomerase from Pyrobaculum aerophilum (PaPGI/PMI) has been determined in native form at 1.16-A resolution and in complex with the enzyme inhibitor 5-phosphoarabinonate at 1.45-A resolution. The similarity of its fold, with the inner core structure of PGIs from eubacterial and eukaryotic sources, confirms this enzyme as a member of the PGI superfamily. The almost total conservation of amino acids in the active site, including the glutamate base catalyst, shows that PaPGI/PMI uses the same catalytic mechanisms for both ring opening and isomerization for the interconversion of glucose 6-phosphate (Glc-6-P) to fructose 6-phosphate (Fru-6-P). The lack of structural differences between native and inhibitor-bound enzymes suggests this activity occurs without any of the conformational changes that are the hallmark of the well characterized PGI family. The lack of a suitable second base in the active site of PaPGI/PMI argues against a PMI mechanism involving a trans-enediol intermediate. Instead, PMI activity may be the result of additional space in the active site imparted by a threonine, in place of a glutamine in other PGI enzymes, which could permit rotation of the C-2-C-3 bond of mannose 6-phosphate.