Publications

• 2-Carboxyquinoxalines kill Mycobacterium tuberculosis through noncovalent inhibition of DprE1.

  Neres J., Hartkoorn R.C., Chiarelli L.R., Gadupudi R., Pasca M.R., Mori G., Venturelli A., Savina S., Makarov V., Kolly G.S., Molteni E., Binda C., Dhar N., Ferrari S., Brodin P., Delorme V., Landry V., de Jesus Lopes Ribeiro A.L., Farina D., Saxena P., Pojer F., Carta A., Luciani R., Porta A., Zanoni G., De Rossi E., Costi M.P., Riccardi G., Cole S.T.

  Phenotypic screening of a quinoxaline library against replicating Mycobacterium tuberculosis led to the identification of lead compound Ty38c (3-((4-methoxybenzyl)amino)-6-(trifluoromethyl)quinoxaline-2-carboxylic acid). With an MIC99 and MBC of 3.1 μM, Ty38c is bactericidal and active against int ... >> More

  Phenotypic screening of a quinoxaline library against replicating Mycobacterium tuberculosis led to the identification of lead compound Ty38c (3-((4-methoxybenzyl)amino)-6-(trifluoromethyl)quinoxaline-2-carboxylic acid). With an MIC99 and MBC of 3.1 μM, Ty38c is bactericidal and active against intracellular bacteria. To investigate its mechanism of action, we isolated mutants resistant to Ty38c and sequenced their genomes. Mutations were found in rv3405c, coding for the transcriptional repressor of the divergently expressed rv3406 gene. Biochemical studies clearly showed that Rv3406 decarboxylates Ty38c into its inactive keto metabolite. The actual target was then identified by isolating Ty38c-resistant mutants of an M. tuberculosis strain lacking rv3406. Here, mutations were found in dprE1, encoding the decaprenylphosphoryl-d-ribose oxidase DprE1, essential for biogenesis of the mycobacterial cell wall. Genetics, biochemical validation, and X-ray crystallography revealed Ty38c to be a noncovalent, noncompetitive DprE1 inhibitor. Structure-activity relationship studies generated a family of DprE1 inhibitors with a range of IC50's and bactericidal activity. Co-crystal structures of DprE1 in complex with eight different quinoxaline analogs provided a high-resolution interaction map of the active site of this extremely vulnerable target in M. tuberculosis. << Less

  ACS Chem. Biol. 10:705-714(2015) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization of a sulfur-regulated oxygenative alkylsulfatase from Pseudomonas putida S-313.

  Kahnert A., Kertesz M.A.

  The atsK gene of Pseudomonas putida S-313 was required for growth with alkyl sulfate esters as sulfur source. The AtsK protein was overexpressed in Escherichia coli and purified to homogeneity. Sequence analysis revealed that AtsK was closely related to E. coli taurine dioxygenase (38% amino acid ... >> More

  The atsK gene of Pseudomonas putida S-313 was required for growth with alkyl sulfate esters as sulfur source. The AtsK protein was overexpressed in Escherichia coli and purified to homogeneity. Sequence analysis revealed that AtsK was closely related to E. coli taurine dioxygenase (38% amino acid identity). The AtsK protein catalyzed the alpha-ketoglutarate-dependent cleavage of a range of alkyl sulfate esters, with chain lengths ranging from C(4) to C(12), required oxygen and Fe(2+) for activity and released succinate, sulfate, and the corresponding aldehyde as products. Enzyme activity was optimal at pH 7 and was strongly stimulated by ascorbate. Unlike most other characterized alpha-ketoglutarate-dependent dioxygenases, AtsK accepted a range of alpha-keto acids as co-substrates, including alpha-ketoglutarate (K(m) 140 microm), alpha-ketoadipate, alpha-ketovalerate, and alpha-ketooctanoate. The measured K(m) values for hexyl sulfate and SDS were 40 and 34 microm, respectively. The apparent M(r) of the purified enzyme of 121,000 was consistent with a homotetrameric structure, which is unusual for this enzyme superfamily, members of which are usually monomeric or dimeric. The properties and amino acid sequence of the AtsK enzyme thus define it as an unusual oxygenolytic alkylsulfatase and a novel member of the alpha-ketoglutarate-dependent dioxygenase family. << Less

  J. Biol. Chem. 275:31661-31667(2000) [PubMed] [EuropePMC]

  This publication is cited by 9 other entries.

• Mycobacterium tuberculosis Rv3406 is a type II alkyl sulfatase capable of sulfate scavenging.

  Sogi K.M., Gartner Z.J., Breidenbach M.A., Appel M.J., Schelle M.W., Bertozzi C.R.

  The genome of Mycobacterium tuberculosis (Mtb) encodes nine putative sulfatases, none of which have a known function or substrate. Here, we characterize Mtb's single putative type II sulfatase, Rv3406, as a non-heme iron (II) and α-ketoglutarate-dependent dioxygenase that catalyzes the oxidation a ... >> More

  The genome of Mycobacterium tuberculosis (Mtb) encodes nine putative sulfatases, none of which have a known function or substrate. Here, we characterize Mtb's single putative type II sulfatase, Rv3406, as a non-heme iron (II) and α-ketoglutarate-dependent dioxygenase that catalyzes the oxidation and subsequent cleavage of alkyl sulfate esters. Rv3406 was identified based on its homology to the alkyl sulfatase AtsK from Pseudomonas putida. Using an in vitro biochemical assay, we confirmed that Rv3406 is a sulfatase with a preference for alkyl sulfate substrates similar to those processed by AtsK. We determined the crystal structure of the apo Rv3406 sulfatase at 2.5 Å. The active site residues of Rv3406 and AtsK are essentially superimposable, suggesting that the two sulfatases share the same catalytic mechanism. Finally, we generated an Rv3406 mutant (Δrv3406) in Mtb to study the sulfatase's role in sulfate scavenging. The Δrv3406 strain did not replicate in minimal media with 2-ethyl hexyl sulfate as the sole sulfur source, in contrast to wild type Mtb or the complemented strain. We conclude that Rv3406 is an iron and α-ketoglutarate-dependent sulfate ester dioxygenase that has unique substrate specificity that is likely distinct from other Mtb sulfatases. << Less

  PLoS ONE 8:E65080-E65080(2013) [PubMed] [EuropePMC]

  This publication is cited by 9 other entries.