Complementation of a metK-deficient E. coli strain with heterologous AdoMet synthetase genes.

S-adenosyl-l-methionine (AdoMet) is an essential metabolite, playing a wide variety of metabolic roles. The enzyme that produces AdoMet from l-methionine and ATP (methionine adenosyltransferase, MAT) is thus an attractive target for anti-cancer and antimicrobial agents. It would be very useful to have a system that allows rapid identification of species-specific inhibitors of this essential enzyme. A previously generated E. coli strain, lacking MAT (∆metK) but containing a heterologous AdoMet transporter, was successfully complemented with heterologous metK genes from several bacterial pathogens, as well as with MAT genes from a fungal pathogen and Homo sapiens. The nine tested genes, which vary in both sequence and kinetic properties, all complemented strain MOB1490 well in rich medium. When these strains were grown in glucose minimal medium, growth delays or defects were observed with some specific metK genes, defects that were dramatically reduced if l-methionine was added to the medium.

Correction of a genetic deficiency in pantothenate kinase 1 using phosphopantothenate replacement therapy.

Coenzyme A (CoA) is a ubiquitous cofactor involved in numerous essential biochemical transformations, and along with its thioesters is a key regulator of intermediary metabolism. Pantothenate (vitamin B5) phosphorylation by pantothenate kinase (PanK) is thought to control the rate of CoA production. Pantothenate kinase associated neurodegeneration is a hereditary disease that arises from mutations that inactivate the human PANK2 gene. Aryl phosphoramidate phosphopantothenate derivatives were prepared to test the feasibility of using phosphopantothenate replacement therapy to bypass the genetic deficiency in the Pank1(-/-) mouse model. The efficacies of candidate compounds were first compared by measuring the ability to increase CoA levels in Pank1(-/-) mouse embryo fibroblasts. Administration of selected candidate compounds to Pank1(-/-) mice corrected their deficiency in hepatic CoA. The PanK bypass was confirmed by the incorporation of intact phosphopantothenate into CoA using triple-isotopically labeled compound. These results provide strong support for PanK as a master regulator of intracellular CoA and illustrate the feasibility of employing PanK bypass therapy to restore CoA levels in genetically deficient mice.

Physiological roles of the pantothenate kinases.

CoA (coenzyme A) is an essential cofactor that is involved in many metabolic processes. CoA is derived from pantothenate in five biosynthetic reactions. The CoA biosynthetic pathway is regulated by PanKs (pantothenate kinases) and four active isoforms are expressed in mammals. The critical physiological functions of the PanKs are revealed by systematic deletion of the Pank genes in mice.

Enhanced brain distribution of modified aspartoacylase.

Canavan disease is a fatal neurological disorder caused by defects in the gene that produces the enzyme aspartoacylase. Enzyme replacement therapy can potentially be used to overcome these defects if a stable enzyme form that can gain access to the appropriate neural cells can be produced. Achieving the proper cellular targeting requires a modified form of aspartoacylase that can traverse the blood-brain barrier. A PEGylated form of aspartoacylase that shows dramatic enhancement in brain tissue access and distribution has been produced. While the mechanism of transport has not yet been established, this modified enzyme is significantly less immunogenic than unmodified aspartoacylase. These improved properties set the stage for more extensive enzyme replacement trials as a possible treatment strategy.

Structure of an unusual S-adenosylmethionine synthetase from Campylobacter jejuni.

S-Adenosylmethionine (AdoMet) participates in a wide range of methylation and other group-transfer reactions and also serves as the precursor for two groups of quorum-sensing molecules that function as regulators of the production of virulence factors in Gram-negative bacteria. The synthesis of AdoMet is catalyzed by AdoMet synthetases (MATs), a ubiquitous family of enzymes found in species ranging from microorganisms to mammals. The AdoMet synthetase from the bacterium Campylobacter jejuni (cjMAT) is an outlier among this homologous enzyme family, with lower sequence identity, numerous insertions and substitutions, and higher catalytic activity compared with other bacterial MATs. Alterations in the structure of this enzyme provide an explanation for its unusual dimeric quaternary structure relative to the other MATs. Taken together with several active-site substitutions, this new structure provides insights into its improved kinetic properties with alternative substrates.

Alternative substrates selective for S-adenosylmethionine synthetases from pathogenic bacteria.

S-adenosyl-l-methionine (AdoMet) synthetase catalyzes the production of AdoMet, the major biological methyl donor and source of methylene, amino, ribosyl, and aminopropyl groups in the metabolism of all known organism. In addition to these essential functions, AdoMet can also serve as the precursor for two different families of quorum sensing molecules that trigger virulence in Gram-negative human pathogenic bacteria. The enzyme responsible for AdoMet biosynthesis has been cloned, expressed and purified from several of these infectious bacteria. AdoMet synthetase (MAT) from Neisseria meningitidis shows similar kinetic parameters to the previously characterized Escherichia coli enzyme, while the Pseudomonas aeruginosa enzyme has a decreased catalytic efficiency for its MgATP substrate. In contrast, the more distantly related MAT from Campylobacter jejuni has an altered quaternary structure and possesses a higher catalytic turnover than the more closely related family members. Methionine analogs have been examined to delineate the substrate specificity of these enzyme forms, and several alternative substrates have been identified with the potential to block quorum sensing while still serving as precursors for essential methyl donation and radical generation reactions.

Relationship between enzyme properties and disease progression in Canavan disease.

Canavan disease (CD) is a fatal neurological disorder caused by defects in the gene that encodes for a critical metabolic enzyme. The enzyme aspartoacylase catalyzes the deacetylation of N-acetylaspartate to produce acetate required for fatty acid biosynthesis in the brain. The loss of aspartoacylase activity leads to the demyelination and disrupted brain development that is found in CD patients. Sixteen different clinical mutants of aspartoacylase have been cloned, expressed and purified to examine their properties and the relationship between enzyme properties and disease phenotype. In contrast to numerous cell culture studies that reported virtually complete loss of function, each of these purified mutant enzymes was found to have measureable catalytic activity. However, the activities of these mutants are diminished, by as little as three-fold to greater than 100-fold when compared to the native enzyme. Many of these mutated enzyme forms show decreased thermal stability and an increased propensity for denaturation upon exposure to urea, but only four of the 16 mutants examined showed both diminished thermal and diminished conformational stability. Significantly, each of these lower stability mutants are responsible for the more severe phenotypes of CD, while patients with milder forms of CD have aspartoacylase mutants with generally high catalytic activity and with either good thermal or good conformational stability. These results suggest that the loss of catalytic function and the accumulation of N-acetylaspartate in Canavan disease is at least partially a consequence of the decreased protein stability caused by these mutations.

Modification of aspartoacylase for potential use in enzyme replacement therapy for the treatment of Canavan disease.

Canavan disease is a fatal neurological disease without any effective treatments to slow the relentless progress of this disorder. Enzyme replacement therapy has been used effectively to treat a number of metabolic disorders, but the presence of the blood-brain-barrier presents an additional challenge in the treatment of neurological disorders. Studies have begun with the aim of establishing a treatment protocol that can effectively replace the defective enzyme in Canavan disease patients. The human enzyme, aspartoacylase, has been cloned, expressed and purified, and the surface lysyl groups modified through PEGylation. Fully active modified enzymes were administered to mice that are defective in this enzyme and that show many of the symptoms of Canavan disease. Statistically significant increases in brain enzyme activity levels have been achieved in this animal model, as well as decreases in the elevated substrate levels that mimic those found in Canavan disease patients. These results demonstrate that the modified enzyme is gaining access to the brain and functions to correct this metabolic defect. The stage is now set for a long term study to optimize this enzyme replacement approach for the development of a treatment protocol.