Tackling Critical Catalytic Residues in Helicobacter pylori L-Asparaginase.

Bacterial asparaginases (amidohydrolases, EC 3.5.1.1) are important enzymes in cancer therapy, especially for Acute Lymphoblastic Leukemia. They are tetrameric enzymes able to catalyze the deamination of L-ASN and, to a variable extent, of L-GLN, on which leukemia cells are dependent for survival. In contrast to other known L-asparaginases, Helicobacter pylori CCUG 17874 type II enzyme (HpASNase) is cooperative and has a low affinity towards L-GLN. In this study, some critical amino acids forming the active site of HpASNase (T16, T95 and E289) have been tackled by rational engineering in the attempt to better define their role in catalysis and to achieve a deeper understanding of the peculiar cooperative behavior of this enzyme. Mutations T16E, T95D and T95H led to a complete loss of enzymatic activity. Mutation E289A dramatically reduced the catalytic activity of the enzyme, but increased its thermostability. Interestingly, E289 belongs to a loop that is very variable in L-asparaginases from the structure, sequence and length point of view, and which could be a main determinant of their different catalytic features.

Clinical Severity of PGK1 Deficiency Due To a Novel p.E120K Substitution Is Exacerbated by Co-inheritance of a Subclinical Translocation t(3;14)(q26.33;q12), Disrupting NUBPL Gene.

Carriers of cytogenetically similar, apparently balanced familial chromosome translocations not always exhibit the putative translocation-associated disease phenotype. Additional genetic defects, such as genomic imbalance at breakpoint regions or elsewhere in the genome, have been reported as the most plausible explanation.By means of comprehensive molecular and functional analyses, additional to careful dissection of the t(3;14)(q26.33;q12) breakpoints, we unveil a novel X-linked PGK1 mutation and examine the contribution of these to the extremely severe clinical phenotype characterized by hemolytic anemia and neuromyopathy.The 3q26.33 breakpoint is 40 kb from the 5' region of tetratricopeptide repeat domain 14 gene (TTC14), whereas the 14q12 breakpoint is within IVS6 of nucleotide-binding protein-like gene (NUBPL) that encodes a mitochondrial complex I assembly factor. Disruption of NUBPL in translocation carriers leads to a decrease in the corresponding mRNA accompanied by a decrease in protein level. Exclusion of pathogenic genomic imbalance and reassessment of familial clinical history indicate the existence of an additional causal genetic defect. Consequently, by WES a novel mutation, c.358G>A, p.E120K, in the X-linked phosphoglycerate kinase 1 (PGK1) was identified that segregates with the phenotype. Specific activity, kinetic properties, and thermal stability of this enzyme variant were severely affected. The novel PGK1 mutation is the primary genetic alteration underlying the reported phenotype as the translocation per se only results in a subclinical phenotype. Nevertheless, its co-inheritance presumably exacerbates PGK1-deficient phenotype, most likely due to a synergistic interaction of the affected genes both involved in cell energy supply.

Engineering of Helicobacter pylori L-asparaginase: characterization of two functionally distinct groups of mutants.

Bacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though presenting, along with their therapeutic efficacy, several side effects due to their bacterial origin and, seemingly, to their secondary glutaminase activity. Helicobacter pylori type II L-asparaginase possesses interesting features, among which a reduced catalytic efficiency for L-GLN, compared to the drugs presently used in therapy. In the present study, we describe some enzyme variants with catalytic and in vitro cytotoxic activities different from the wild type enzyme. Particularly, replacements on catalytic threonines (T16D and T95E) deplete the enzyme of both its catalytic activities, once more underlining the essential role of such residues. One serendipitous mutant, M121C/T169M, had a preserved efficiency vs L-asparagine but was completely unable to carry out L-glutamine hydrolysis. Interestingly, this variant did not exert any cytotoxic effect on HL-60 cells. The M121C and T169M single mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme, respectively). Mutant Q63E, endowed with a similar catalytic efficiency versus asparagine and halved glutaminase efficiency with respect to the wild type enzyme, was able to exert a cytotoxic effect comparable to, or higher than, the one of the wild type enzyme when similar asparaginase units were used. These findings may be relevant to determine the role of glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed light on how to engineer the best asparaginase/glutaminase combination for an ever improved, patients-tailored therapy.

Biochemistry of lipolytic enzymes secreted by Penicillium solitum and Cladosporium cladosporioides.

Two distinct extracellular lipases were obtained from Penicillium solitum 194A, isolated from domestic compost, and Cladosporium cladosporioides 194B, isolated from dairy wastewater. These alkaline enzymes had molecular masses of 42 and 30 kDa, respectively. The P. solitum 194A lipase differed in mass from previously reported enzyme, indicating that it is a novel lipase, and indicating that penicillia can secrete lipase isoenzymes. The C. cladosporioides lipase was more active on esters of medium-chain acids, whereas the P. solitum lipase was more active on longer chained substrates. The C. cladosporioides enzyme displayed higher thermal stability than the P. solitum lipase, preserving full activity up to 48 °C and showing a T50 (10 min) of 60 °C. Their different catalytic properties and good protein stability should make these enzymes suitable for biotechnological applications. Furthermore, the combined use of these two fungal strains may prove to be valuable in lipid-rich waste management.

Insights into human phosphoglycerate kinase 1 deficiency as a conformational disease from biochemical, biophysical, and in vitro expression analyses.

Mutations in genes encoding metabolic enzymes are often the cause of inherited diseases. Mutations usually affect the ability of proteins to fold properly, thus leading to enzyme loss of function. In this work, we explored the relationships between protein stability, aggregation, and degradation in vitro and inside cells in a large set of mutants associated with human phosphoglycerate kinase 1 (hPGK1) deficiency. To this end, we studied a third of the pathogenic alleles reported in the literature using expression analyses and biochemical, biophysical, and computational procedures. Our results show that most pathogenic variants studied had an increased tendency to aggregate when expressed in Escherichia coli, well correlating with the denaturation half-lives measured by thermal denaturation in vitro. Further, the most deleterious mutants show reduced stability toward chemical denaturation and proteolysis, supporting a pivotal role of thermodynamic stability in the propensity toward aggregation and proteolysis of pathogenic hPGK1 mutants in vitro and inside cells. Our strategy allowed us to unravel the complex relationships between protein stability, aggregation, and degradation in hPGK1 deficiency, which might be used to understand disease mechanisms in many inborn errors of metabolism. Our results suggest that pharmacological chaperones and protein homeostasis modulators could be considered as good candidates for therapeutic approaches for hPGK1 deficiency.

A strategy for synthesis of pathogenic human immunoglobulin free light chains in E. coli.

Monoclonal immunoglobulin light chains are normally synthesized in excess compared to the heavy chain partners and can be detected in serum and urine ("free" LC). Occasionally free LC are per se cause of organ toxicity, as in free LC-related disorders. In AL amyloidosis, the most common of these conditions, free LC with peculiar biophysical properties related to their primary structure damage target organs and organize in amyloid fibrils. Unlimited availability of well-characterized free LC is instrumental to investigate the toxic effect of these proteins and to study their interactions with targets. We present a straightforward strategy to obtain recombinant monoclonal free LC by using a bacterial system. These proteins, expressed as inclusion bodies, were subjected to solubilization and refolding procedures to recover them in native form. To minimize differences from the circulating natural LC, full-length recombinant LC were expressed, i.e. complete of variable and constant regions, with the original amino acid sequence along the entire protein, and with no purification tags. The strategy was exploited to generate free LC from three AL amyloidosis patients. After purification, recombinant proteins were biochemically characterized and compared to the natural Bence Jones protein isolated from one of the patients. Results showed that the recombinant free LC were properly folded and formed homodimers in solution, similar to the natural Bence Jones protein used for comparison. Furthermore, as proof of pathogenicity, recombinant proteins formed amyloid fibrils in vitro. We believe that the present strategy represents a valuable tool to speed research in free LC-related disorders.

Structural and energetic basis of protein kinetic destabilization in human phosphoglycerate kinase 1 deficiency.

Protein kinetic destabilization is a common feature of many human genetic diseases. Human phosphoglycerate kinase 1 (PGK1) deficiency is a rare genetic disease caused by mutations in the PGK1 protein, which often shows reduced kinetic stability. In this work, we have performed an in-depth characterization of the thermal stability of the wild type and four disease-causing mutants (I47N, L89P, E252A, and T378P) of human PGK1. PGK1 thermal denaturation is a process under kinetic control, and it is described well by a two-state irreversible denaturation model. Kinetic analysis of differential scanning calorimetry profiles shows that the disease-causing mutations decrease PGK1 kinetic stability from ~5-fold (E252A) to ~100000-fold (L89P) compared to that of wild-type PGK1, and in some cases, mutant enzymes are denatured on a time scale of a few minutes at physiological temperature. We show that changes in protein kinetic stability are associated with large differences in enthalpic and entropic contributions to denaturation free energy barriers. It is also shown that the denaturation transition state becomes more nativelike in terms of solvent exposure as the protein is destabilized by mutations (Hammond effect). Unfolding experiments with urea further suggest a scenario in which the thermodynamic stability of PGK1 at least partly determines its kinetic stability. ATP and ADP kinetically stabilize PGK1 enzymes, and kinetic stabilization is nucleotide- and mutant-selective. Overall, our data provide insight into the structural and energetic basis underlying the low kinetic stability displayed by some mutants causing human PGK1 deficiency, which may have important implications for the development of native state kinetic stabilizers for the treatment of this disease.

Protein Stability, Folding and Misfolding in Human PGK1 Deficiency.

Conformational diseases are often caused by mutations, altering protein folding and stability in vivo. We review here our recent work on the effects of mutations on the human phosphoglycerate kinase 1 (hPGK1), with a particular focus on thermodynamics and kinetics of protein folding and misfolding. Expression analyses and in vitro biophysical studies indicate that disease-causing mutations enhance protein aggregation propensity. We found a strong correlation among protein aggregation propensity, thermodynamic stability, cooperativity and dynamics. Comparison of folding and unfolding properties with previous reports in PGKs from other species suggests that hPGK1 is very sensitive to mutations leading to enhance protein aggregation through changes in protein folding cooperativity and the structure of the relevant denaturation transition state for aggregation. Overall, we provide a mechanistic framework for protein misfolding of hPGK1, which is insightful to develop new therapeutic strategies aimed to target native state stability and foldability in hPGK1 deficient patients.

A new variant of phosphoglycerate kinase deficiency (p.I371K) with multiple tissue involvement: molecular and functional characterization.

Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible phosphotransfer reaction from 1,3-bisphosphoglycerate to MgADP, to form 3-phosphoglycerate and MgATP. Two isozymes encoded by distinct genes are present in humans: PGK-1, located on Xq-13.3, encodes a ubiquitous protein of 417 amino acids, whereas PGK-2 is testis-specific. PGK1 deficiency is characterized by mild to severe hemolytic anemia, neurological dysfunctions and myopathy; patients rarely exhibit all three clinical features. Nearly 40 cases have been reported, 27 of them characterized at DNA or protein level, and 20 different mutations were described. Here we report the first Italian case of PGK deficiency characterized at a molecular and biochemical level. The patient presented during infancy with hemolytic anemia, increased CPK values, and respiratory distress; the study of red blood cell enzymes showed a drastic reduction in PGK activity. In adulthood he displayed mild hemolytic anemia, mental retardation and severe myopathy. PGK-1 gene sequencing revealed the new missense mutation c.1112T>A (p.Ile371Lys). The mutation was not found among 100 normal alleles, and even if located in the third to the last nucleotide of exon 9, it did not alter mRNA splicing. The p.Ile371Lys mutation falls in a conserved region of the enzyme, near the nucleotide binding site. The mutant enzyme shows reduced catalytic rates toward both substrates (apparent k(cat) values, 12-fold lower than wild-type) and a decreased affinity toward MgATP (apparent K(m), 6-fold higher than wild-type). Moreover, it lost half of activity after nearly 9-min incubation at 45°C, a temperature that did not affect the wild-type enzyme (t(1/2)>1 h). The possible compensatory expression of PGK2 isoenzyme was investigated in the proband and in the heterozygote healthy sisters, and found to be absent. Therefore, the highly perturbed catalytic properties of the new variant p.Ile371Lys, combined with protein instability, account for the PGK deficiency found in the patient and correlate with the clinical expression of the disease.

Molecular insights on pathogenic effects of mutations causing phosphoglycerate kinase deficiency.

Phosphoglycerate kinase (PGK) catalyzes an important ATP-generating step in glycolysis. PGK1 deficiency is an uncommon X-linked inherited disorder, generally characterized by various combinations of non-spherocytic hemolytic anemia, neurological dysfunctions, and myopathies. Patients rarely exhibit all three clinical features. To provide a molecular framework to the different pathological manifestations, all known mutations were reviewed and 16 mutant enzymes, obtained as recombinant forms, were functionally and structurally characterized. Most mutations heavily affect thermal stability and to a different extent catalytic efficiency, in line with the remarkably low PGK activity clinically observed in the patients. Mutations grossly impairing protein stability, but moderately affecting kinetic properties (p.I47N, p.L89P, p.C316R, p.S320N, and p.A354P) present the most homogeneous correlation with the clinical phenotype. Patients carrying these mutations display hemolytic anemia and neurological disorders, and,except for p.A354P variant, no myopaty. Variants highly perturbed in both catalytic efficiency (p.G158V, p.D164V, p.K191del, D285V, p.D315N, and p.T378P) and heat stability (all, but p.T378P) result to be mainly associated with myopathy alone. Finally, mutations faintly affecting molecular properties (p.R206P, p.E252A, p.I253T, p.V266M, and p.D268N) correlate with a wide spectrum of clinical symptoms. These are the first studies that correlate the clinical symptoms with the molecular properties of the mutant enzymes. All findings indicate that the different clinical manifestations associated with PGK1 deficiency chiefly depend on the distinctive type of perturbations caused by mutations in the PGK1 gene, highlighting the need for determination of the molecular properties of PGK variants to assist in prognosis and genetic counseling. However, the clinical symptoms can not be understood only on the bases of molecular properties of the mutant enzyme. Different (environmental, metabolic, genetic and/or epigenetic) intervening factors can contribute toward the expression of PGK deficient clinical phenotypes.

Expanding targets for a metabolic therapy of cancer: L-asparaginase.

The antitumour enzyme L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1, ASNase), which catalyses the deamidation of L-asparagine (Asn) to L-aspartic acid and ammonia, has been used for many years in the treatment of acute lymphoblastic leukaemia. Also NK tumours, subtypes of myeloid leukaemias and T-cell lymphomas respond to ASNase, and ovarian carcinomas and other solid tumours have been proposed as additional targets for ASNase, with a potential role for its glutaminase activity. The increasing attention devoted to the antitumour activity of ASNase prompted us to analyse recent patents specifically concerning this enzyme. Here, we first give an overview of metabolic pathways affected by Asn and Gln depletion and, hence, potential targets of ASNase. We then discuss recent published patents concerning ASNases. In particular, we pay attention to novel ASNases, such as the recently characterised ASNase produced by Helicobacter pylori, and those presenting amino acid substitutions aimed at improving enzymatic activity of the classical Escherichia coli enzyme. We detail modifications, such as natural glycosylation or synthetic conjugation with other molecules, for therapeutic purposes. Finally, we analyse patents concerning biotechnological protocols and strategies applied to production of ASNase as well as to its administration and delivery in organisms.

Analogous mechanisms of resistance to benzothiazinones and dinitrobenzamides in Mycobacterium smegmatis.

Tuberculosis is still a leading cause of death worldwide. The selection and spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) is a severe public health problem. Recently, two different classes of chemical series, the benzothiazinones (BTZ) and the dinitrobenzamide (DNB) derivatives have been found to be highly active against M. tuberculosis, including XDR-TB strains. The target of BTZs is DprE1 protein which works in concert with DprE2 to form the heteromeric decaprenylphosphoryl-ß-D-ribose 2'-epimerase, involved in Decaprenyl-Phospho-Arabinose (DPA) biosynthesis. Interestingly, it has been shown that the DNBs block the same pathway thus suggesting that both drugs could share the same target. Moreover, in Mycobacterium smegmatis the overexpression of the NfnB nitroreductase led to the inactivation of the BTZs by reduction of a critical nitro-group to an amino-group. In this work several spontaneous M. smegmatis mutants resistant to DNBs were isolated. Sixteen mutants, showing high levels of DNB resistance, exhibited a mutation in the Cys394 of DprE1. Using fluorescence titration and mass spectrometry it has been possible to monitor the binding between DprE1 and DNBs, achieving direct evidence that MSMEG_6382 is the cellular target of DNBs in mycobacteria. Additionally, M. smegmatis mutants having low levels of resistance to DNBs harbor various mutations in MSMEG_6503 gene encoding the transcriptional repressor of the nitroreductase NfnB. By LC/MS analysis it has been demonstrated that NfnB is responsible for DNB inactivation. Taken together, our data demonstrate that both DNB and BTZ drugs share common resistance mechanisms in M. smegmatis.

Cell-cycle inhibition by Helicobacter pylori L-asparaginase.

Helicobacter pylori (H. pylori) is a major human pathogen causing chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. One of the mechanisms whereby it induces damage depends on its interference with proliferation of host tissues. We here describe the discovery of a novel bacterial factor able to inhibit the cell-cycle of exposed cells, both of gastric and non-gastric origin. An integrated approach was adopted to isolate and characterise the molecule from the bacterial culture filtrate produced in a protein-free medium: size-exclusion chromatography, non-reducing gel electrophoresis, mass spectrometry, mutant analysis, recombinant protein expression and enzymatic assays. L-asparaginase was identified as the factor responsible for cell-cycle inhibition of fibroblasts and gastric cell lines. Its effect on cell-cycle was confirmed by inhibitors, a knockout strain and the action of recombinant L-asparaginase on cell lines. Interference with cell-cycle in vitro depended on cell genotype and was related to the expression levels of the concurrent enzyme asparagine synthetase. Bacterial subcellular distribution of L-asparaginase was also analysed along with its immunogenicity. H. pylori L-asparaginase is a novel antigen that functions as a cell-cycle inhibitor of fibroblasts and gastric cell lines. We give evidence supporting a role in the pathogenesis of H. pylori-related diseases and discuss its potential diagnostic application.

Mycobacterium tuberculosis phosphoribosylpyrophosphate synthetase: biochemical features of a crucial enzyme for mycobacterial cell wall biosynthesis.

The selection and soaring spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) is a severe public health problem. Currently, there is an urgent need for new drugs for tuberculosis treatment, with novel mechanisms of action and, moreover, the necessity to identify new drug targets. Mycobacterial phosphoribosylpyrophosphate synthetase (MtbPRPPase) is a crucial enzyme involved in the biosynthesis of decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Moreover, phosphoribosylpyrophosphate, which is the product of the PRPPase catalyzed reaction, is the precursor for the biosynthesis of nucleotides and of some amino acids such as histidine and tryptophan. In this context, the elucidation of the molecular and functional features of MtbPRPPase is mandatory. MtbPRPPase was obtained as a recombinant form, purified to homogeneity and characterized. According to its hexameric form, substrate specificity and requirement of phosphate for activity, the enzyme proved to belong to the class I of PRPPases. Although the sulfate mimicked the phosphate, it was less effective and required higher concentrations for the enzyme activation. MtbPRPPase showed hyperbolic response to ribose 5-phosphate, but sigmoidal behaviour towards Mg-ATP. The enzyme resulted to be allosterically activated by Mg(2+) or Mn(2+) and inhibited by Ca(2+) and Cu(2+) but, differently from other characterized PRPPases, it showed a better affinity for the Mn(2+) and Cu(2+) ions, indicating a different cation binding site geometry. Moreover, the enzyme from M. tuberculosis was allosterically inhibited by ADP, but less sensitive to inhibition by GDP. The characterization of M. tuberculosis PRPPase provides the starting point for the development of inhibitors for antitubercular drug design.

Biochemical signatures of doppel protein in human astrocytomas to support prediction in tumor malignancy.

Doppel (Dpl) is a membrane-bound glycoprotein mainly expressed in the testis of adult healthy people. It is generally absent in the central nervous system, but its coding gene sequence is ectopically expressed in astrocytoma specimens and in derived cell lines. In this paper, we investigated the expression and the biochemical features of Dpl in a panel of 49 astrocytoma specimens of different WHO malignancy grades. As a result, Dpl was expressed in the majority of the investigated specimens (86%), also including low grade samples. Importantly, Dpl exhibited different cellular localizations and altered glycan moieties composition, depending on the tumor grade. Most low-grade astrocytomas (83%) showed a membrane-bound Dpl, like human healthy testis tissue, whereas the majority of high-grade astrocytomas (75%) displayed a cytosolic Dpl. Deglycosylation studies with N-glycosidase F and/or neuraminidase highlighted defective glycan moieties and an unexpected loss of sialic acid. To find associations between glial tumor progression and Dpl biochemical features, predictive bioinformatics approaches were produced. In particular, Decision tree and Nomogram analysis showed well-defined Dpl-based criteria that separately clustered low-and high-grade astrocytomas. Taken together, these findings show that in astrocytomas, Dpl undergoes different molecular processes that might constitute additional helpful tools to characterize the glial tumor progression.

Inosine monophosphate dehydrogenase variability in renal transplant patients on long-term mycophenolate mofetil therapy.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: * Mycophenolic acid (MPA) is a potent, selective and reversible inhibitor of inosine 5'-monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme for de novo guanosine triphosphate biosynthesis. * The large IMPDH interindividual variability could be responsible for the differences in therapeutic effects and side-effects observed with MPA. * Induction of IMPDH activity has been observed in whole blood during immunosuppressive therapy. WHAT THIS STUDY ADDS: * Our data were acquired in long-term mycophenolate mofetil-treated renal transplant recipients on different combinations of immunosuppressive agents (ciclosporin, tacrolimus, sirolimus) and with different treatment duration (up to 8.8 years post transplant). * The increasing trend in IMPDH activity that we observed throughout our 12-month observation period was significantly higher in rejecting than in nonrejecting subjects. AIMS: Long-term mycophenolate mofetil (MMF) therapy may induce inosine 5'-monophosphate dehydrogenase (IMPDH) activity in peripheral blood mononuclear cells (PBMCs), thus decreasing MMF immunosuppressive properties. Pharmacodynamic monitoring was used to investigate whether biological activity is altered after long-term therapy. METHODS: IMPDH activity was measured in PBMC samples from 54 stable kidney transplant patients, already on MMF (for at least 3 months), before (t(0)) and 2 h after (t(2)) MMF morning dose administration; levels were monitored for up to 15 months, together with total mycophenolic acid (MPA) and free MPA concentrations. RESULTS: During the 15 months' monitoring, t(0) IMPDH activity in transplant recipients increased from 5.9 +/- 3.7 nmol h(-1) mg(-1)[95% confidence interval (CI) 4.9, 6.9] to 9.0 +/- 3.9 nmol h(-1) mg(-1) (95% CI 7.2, 10.8), with an intra- and interpatient variability of 28% and 42%. Five patients experienced acute rejection during the follow-up: t(0) IMPDH activity was increased during rejection vs. nonrejection, and the trend was significantly higher in rejecting than in nonrejecting subjects for the whole monitoring period. CONCLUSIONS: Even though a correlation has been found between IMPDH activity and rejection, its efficacy as a predictive tool in long-term transplant outcomes may be affected by high interpatient variability; on the other hand, continuous monitoring of the IMPDH trend could make an effective prognostic parameter of rejection. Other trials also including pre-transplant data on both IMPDH expression and activity are warranted to better assess their role as biomarkers for MPA effect in clinical practice.

Helicobacter pyloril-asparaginase: a promising chemotherapeutic agent.

Bacterial L-asparaginases are amidohydrolases that catalyse the conversion of L-asparagine to L-aspartate and ammonia and are used as anti-cancer drugs. The current members of this class of drugs have several toxic side effects mainly due to their associated glutaminase activity. In the present study, we report the molecular cloning, biochemical characterisation and in vitro cytotoxicity of a novel L-asparaginase from the pathogenic strain Helicobacter pylori CCUG 17874. The recombinant enzyme showed a strong preference for L-asparagine over L-glutamine and, in contrast to most L-asparaginases, it exhibited a sigmoidal behaviour towards L-glutamine. The enzyme preserved full activity after 2 h incubation at 45 degrees C. In vitro cytotoxicity assays revealed that different cell lines displayed a variable sensitivity towards the enzyme, AGS and MKN28 gastric epithelial cells being the most affected. These findings may be relevant both for the interpretation of the mechanisms underlying H. pylori associated diseases and for biomedical applications.

Molecular basis of pyrimidine 5'-nucleotidase deficiency caused by 3 newly identified missense mutations (c.187T>C, c.469G>C and c.740T>C) and a tabulation of known mutations.

Hereditary pyrimidine 5'-nucleotidase deficiency is the most frequent enzymopathy of red blood cell nucleotide metabolism that causes hereditary non-spherocytic hemolytic anemia. The disease is usually characterized by mild-to-moderate hemolytic anemia, reticulocytosis and hyperbilirubinemia. To date, diagnosis ultimately depends upon demonstration of a reduced level of pyrimidine 5'-nucleotidase type-I (P5'N-1) activity in red cells and detection of mutations in the P5'N-1 gene. To unravel the causes of the P5'N deficiency and to obtain data for a definitive diagnosis three newly described missense mutations (c.187T>C, c.469G>C and c.740T>C) identified in patients with hemolytic anemia have been characterized at protein level. The mutant enzymes (C63R, G157R and I247T) were obtained as recombinant forms and purified to homogeneity. The enzymes were altered, although to a different extent, in both thermal stability and catalytic efficiency. The catalytic efficiency of all mutants was reduced especially towards UMP (up to more than 200 times), owing to the increased Km values (approximately, 10-25 times higher). The G157R enzyme was severely heat unstable and lost half of its activity after about 23 min of incubation at 37 degrees C. At higher temperature C63R and I247T mutants as well were less stable than the wild-type enzyme. Therefore, although the mutations targeted different regions of the P5'N-1 structure, they produced similar effects on the molecular properties of the enzyme. Thus, all affected amino acids are functionally and structurally important for preserving the enzyme activity during the red cell life span.

Erythrocyte adenylate kinase deficiency: characterization of recombinant mutant forms and relationship with nonspherocytic hemolytic anemia.

OBJECTIVE: Red cell adenylate kinase (AK) deficiency is a rare hereditary erythroenzymopathy associated with moderate to severe nonspherocytic hemolytic anemia and, in some cases, with mental retardation and psychomotor impairment. To date, diagnosis of AK deficiency depends upon demonstration of low enzyme activity in red blood cells and detection of mutations in AK1 gene. To investigate the molecular bases of the AK deficiency, we characterized five variants of AK1 isoenzyme-bearing mutations (118G>A, 190G>A, 382C>T, 418-420del, and 491A>G) found in AK-deficient patients with chronic hemolytic anemia. MATERIALS AND METHODS: The complete AK1 cDNA was obtained by standard procedures and using as template the reticulocyte RNA. The cDNA was cloned in a plasmid vector and the enzyme was expressed in Escherichia coli BL21(DE3)pLysS, and purified by standard protocols to homogeneity. DNA mutants bearing point mutations were obtained from the cloned wild-type cDNA using standard methods of site-directed mutagenesis, whereas the DNA mutant with deletion of codon 140 was obtained by a two-step method. RESULTS: Four mutant enzymes (Gly40Arg, Gly64Arg, Arg128Trp, Asp140del) were severely affected in activity, displaying a catalytic efficiency of four orders of magnitude lower than the wild-type; one (Tyr164Cys) was grossly perturbed in protein stability. CONCLUSIONS: The altered properties displayed by the mutant enzymes support the cause-effect relationship between AK1 mutations and hemolytic anemia.