Molecular characterization of AmiC, a positive regulator in acetamidase operon of Mycobacterium smegmatis

Venkatesan, Arunkumar; Palaniyandi, Kannan; Narayanan, Sujatha

doi:10.1007/s12192-017-0861-2

Cited by 3 publications

(2 citation statements)

References 26 publications

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“…Notably, no other clusters within the SBP_bac_5 family were associated with a FmdA_AmdA protein, and the structure of these amidase-associated operons was clearly distinct from previously well-studied amidase operons such as the Pseudomonas aeruginosa amidase operon, , Rhodococcus sp. R312 amidase operon, and Mycobacterium smegmatis acetamidase operon − (Figure S3). This marked difference prompted further investigation into this distinct set of ABC transporter-associated proteins.…”

Section: Resultsmentioning

confidence: 99%

Identification and Characterization of a Bacterial Periplasmic Solute Binding Protein That Binds l-Amino Acid Amides

Smith,

Frkic,

Rahman

et al. 2024

Biochemistry

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Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from Mesorhizobium sp. A09 (MeAmi_SBP) and show that MeAmi_SBP binds L-amino acid amides but not the corresponding L-amino acids. An X-ray crystal structure of MeAmi_SBP bound to L-serinamide highlights the residues that impart distinct specificity for L-amino acid amides and reveals a structural Ca 2+ binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca 2+ binding are conserved among the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake, and metabolism of L-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that MeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase−glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with L-amino acid amides and bacterial nitrogen utilization.

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Section: Resultsmentioning

confidence: 99%

Identification and Characterization of a Bacterial Periplasmic Solute Binding Protein That Binds l-Amino Acid Amides

Smith,

Frkic,

Rahman

et al. 2024

Biochemistry

View full text Add to dashboard Cite

show abstract

“…Notably, no other clusters within the SBP_bac_5 family were associated with a FmdA_AmdA protein and the structure of these amidase-associated operons was clearly distinct from previously well-studied amidase operons such as the Pseudomonas aeruginosa amidase operon (35,36), Rhodococcus sp. R312 amidase operon (37) and Mycobacterium smegmatis acetamidase operon (38)(39)(40) (Supplementary Figure 1). This marked difference prompted further investigation into this distinct set of ABC transporter-associated proteins.…”

Section: Identification Of a Group Of Uncharacterized Sbps From Amida...mentioning

confidence: 99%

Identification and characterization of a bacterial periplasmic solute binding protein that binds L-amino acid amides

Smith,

Frkic,

Rahman

et al. 2024

Preprint

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Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP fromMesorhizobiumsp. A09 (MeAmi_SBP) and show thatMeAmi_SBP bindsl-amino acid amides but not the correspondingl-amino acids. An X-ray crystal structure ofMeAmi_SBP bound tol-serinamide highlights the residues that impart distinct specificity forl-amino acid amides and reveals a structural Ca2+binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca2+binding are conserved amongst the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake and metabolism ofl-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose thatMeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions withl-amino acid amides and bacterial nitrogen utilization.

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Identification and optimization of pyridine carboxamide-based scaffold as a drug lead for Mycobacterium tuberculosis

Singh,

Kumar,

Sharma

et al. 2024

Antimicrob Agents Chemother

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New drugs with novel mechanisms of action are urgently needed to tackle the issue of drug-resistant tuberculosis. Here, we have performed phenotypic screening using the Pathogen Box library obtained from the Medicines for Malaria Venture against Mycobacterium tuberculosis in vitro . We have identified a pyridine carboxamide derivative, MMV687254, as a promising hit. This molecule is specifically active against M. tuberculosis and Mycobacterium bovis Bacillus Calmette-Guérin ( M. bovis BCG) but inactive against Enterococcus faecalis , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumanii , Pseudomonas aeruginosa , and Escherichia coli pathogens. We demonstrate that MMV687254 inhibits M. tuberculosis growth in liquid cultures in a bacteriostatic manner. Surprisingly, MMV687254 was as active as isoniazid in macrophages and inhibited M. tuberculosis growth in a bactericidal manner. Mechanistic studies revealed that MMV687254 is a prodrug and that its anti-mycobacterial activity requires AmiC - dependent hydrolysis. We further demonstrate that MMV687254 inhibits M. tuberculosis growth in macrophages by inducing autophagy. In the present study, we have also carried out a detailed structure-activity relationship study and identified a promising novel lead candidate. The identified novel series of compounds also showed activity against drug-resistant M. bovis BCG and M. tuberculosis clinical strains. Finally, we demonstrate that in contrast to MMV687254, the lead molecule was able to inhibit M. tuberculosis growth in a chronic mouse model of infection. Taken together, we have identified a novel lead molecule with a dual mechanism of action that can be further optimized to design more potent anti-tubercular agents.

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Molecular characterization of AmiC, a positive regulator in acetamidase operon of Mycobacterium smegmatis

Cited by 3 publications

References 26 publications

Identification and Characterization of a Bacterial Periplasmic Solute Binding Protein That Binds l-Amino Acid Amides

Identification and Characterization of a Bacterial Periplasmic Solute Binding Protein That Binds l-Amino Acid Amides

Identification and characterization of a bacterial periplasmic solute binding protein that binds L-amino acid amides

Identification and optimization of pyridine carboxamide-based scaffold as a drug lead for Mycobacterium tuberculosis

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