Tight binding enantiomers of pre-clinical drug candidates.

MTDIA is a picomolar transition state analogue inhibitor of human methylthioadenosine phosphorylase and a femtomolar inhibitor of Escherichia coli methylthioadenosine nucleosidase. MTDIA has proven to be a non-toxic, orally available pre-clinical drug candidate with remarkable anti-tumour activity against a variety of human cancers in mouse xenografts. The structurally similar compound MTDIH is a potent inhibitor of human and malarial purine nucleoside phosphorylase (PNP) as well as the newly discovered enzyme, methylthioinosine phosphorylase, isolated from Pseudomonas aeruginosa.

Determinants of the CmoB carboxymethyl transferase utilized for selective tRNA wobble modification.

Enzyme-mediated modifications at the wobble position of tRNAs are essential for the translation of the genetic code. We report the genetic, biochemical and structural characterization of CmoB, the enzyme that recognizes the unique metabolite carboxy-S-adenosine-L-methionine (Cx-SAM) and catalyzes a carboxymethyl transfer reaction resulting in formation of 5-oxyacetyluridine at the wobble position of tRNAs. CmoB is distinctive in that it is the only known member of the SAM-dependent methyltransferase (SDMT) superfamily that utilizes a naturally occurring SAM analog as the alkyl donor to fulfill a biologically meaningful function.

Active site and remote contributions to catalysis in methylthioadenosine nucleosidases.

5'-Methylthioadenosine/S-adenosyl-l-homocysteine nucleosidases (MTANs) catalyze the hydrolysis of 5'-methylthioadenosine to adenine and 5-methylthioribose. The amino acid sequences of the MTANs from Vibrio cholerae (VcMTAN) and Escherichia coli (EcMTAN) are 60% identical and 75% similar. Protein structure folds and kinetic properties are similar.

Structural and biochemical characterization of Chlamydia trachomatis hypothetical protein CT263 supports that menaquinone synthesis occurs through the futalosine pathway.

The obligate intracellular human pathogen Chlamydia trachomatis is the etiological agent of blinding trachoma and sexually transmitted disease. Genomic sequencing of Chlamydia indicated this medically important bacterium was not exclusively dependent on the host cell for energy. In order for the electron transport chain to function, electron shuttling between membrane-embedded complexes requires lipid-soluble quinones (e.

Catalytic site cooperativity in dimeric methylthioadenosine nucleosidase.

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidases (MTANs) are bacterial enzymes that catalyze hydrolysis of the N-ribosidic bonds of 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) to form adenine and 5-thioribosyl groups. MTANs are involved in AI-1 and AI-2 bacterial quorum sensing and the unusual futalosine-based menaquinone synthetic pathway in Streptomyces, Helicobacter, and Campylobacter species. Crystal structures show MTANs to be homodimers with two catalytic sites near the dimer interface.

Salmonella enterica MTAN at 1.36 Å resolution: a structure-based design of tailored transition state analogs.

Accumulation of 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) in bacteria disrupts the S-adenosylmethionine pool to alter biological methylations, synthesis of polyamines, and production of quorum-sensing molecules. Bacterial metabolism of MTA and SAH depends on MTA/SAH nucleosidase (MTAN), an enzyme not present in humans and a target for quorum sensing because MTAN activity is essential for synthesis of autoinducer-2 molecules. Crystals of Salmonella enterica MTAN with product and transition state analogs of MTA and SAH explain the structural contacts causing pM binding affinity for the inhibitor and reveal a "water-wire" channel for the catalytic nucleophile.

Femtomolar inhibitors bind to 5'-methylthioadenosine nucleosidases with favorable enthalpy and entropy.

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the hydrolytic cleavage of adenine from methylthioadenosine (MTA). Inhibitor design and synthesis informed by transition state analysis have developed femtomolar inhibitors for MTANs, among the most powerful known noncovalent enzyme inhibitors. Thermodynamic analyses of the inhibitor binding reveals a combination of highly favorable contributions from enthalpic (-24.

A complex of methylthioadenosine/S-adenosylhomocysteine nucleosidase, transition state analogue, and nucleophilic water identified by mass spectrometry.

An enzyme-stabilized nucleophilic water molecule has been implicated at the transition state of Escherichia coli methylthioadenosine nucleosidase (EcMTAN) by transition state analysis and crystallography. We analyzed the EcMTAN mass in complex with a femtomolar transition state analogue to determine whether the inhibitor and nucleophilic water could be detected in the gas phase. EcMTAN-inhibitor and EcMTAN-inhibitor-nucleophilic water complexes were identified by high-resolution mass spectrometry under nondenaturing conditions.