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.

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.