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Biochemical Investigation of Rv3404c from Mycobacterium tuberculosis.

ABSTRACT: The causative agent of tuberculosis, Mycobacterium tuberculosis, is a bacterium with a complex cell wall and a complicated life cycle. The genome of M. tuberculosis contains well over 4000 genes thought to encode proteins. One of these codes for a putative enzyme referred to as Rv3404c, which has attracted research attention as a potential virulence factor for over 12 years. Here we demonstrate that Rv3404c functions as a sugar N-formyltransferase that converts dTDP-4-amino-4,6-dideoxyglucose into dTDP-4-formamido-4,6-dideoxyglucose using N10-formyltetrahydrofolate as the carbon source. Kinetic analyses demonstrate that Rv3404c displays a significant catalytic efficiency of 1.1 × 104 M-1 s-1. In addition, we report the X-ray structure of a ternary complex of Rv3404c solved in the presence of N5-formyltetrahydrofolate and dTDP-4-amino-4,6-dideoxyglucose. The final model of Rv3404c was refined to an overall R-factor of 16.8% at 1.6 Å resolution. The results described herein are especially intriguing given that there have been no published reports of N-formylated sugars associated with M. tuberculosis. The data thus provide a new avenue of research into this fascinating, yet deadly, organism that apparently has been associated with human infection since ancient times.

SUBMITTER: Dunsirn MM

PROVIDER: S-EPMC5644030 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Biochemical Investigation of Rv3404c from Mycobacterium tuberculosis.

Dunsirn Murray M MM Thoden James B JB Gilbert Michel M Holden Hazel M HM

Biochemistry 20170714 29

The causative agent of tuberculosis, Mycobacterium tuberculosis, is a bacterium with a complex cell wall and a complicated life cycle. The genome of M. tuberculosis contains well over 4000 genes thought to encode proteins. One of these codes for a putative enzyme referred to as Rv3404c, which has attracted research attention as a potential virulence factor for over 12 years. Here we demonstrate that Rv3404c functions as a sugar N-formyltransferase that converts dTDP-4-amino-4,6-dideoxyglucose in ...[more]

PMID: 28665588

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Project description:The Mycobacterium tuberculosis genome harbors nine toxin-antitoxin (TA) systems that are members of the mazEF family, unlike other prokaryotes, which have only one or two. Although the overall tertiary folds of MazF toxins are predicted to be similar, it is unclear how they recognize structurally different RNAs and antitoxins with divergent sequence specificity. Here, we have expressed and purified the individual components and complex of the MazEF6 TA system from M. tuberculosis. Size exclusion chromatography-multiangle light scattering (SEC-MALS) was performed to determine the oligomerization status of the toxin, antitoxin, and the complex in different stoichiometric ratios. The relative stabilities of the proteins were determined by nano-differential scanning fluorimetry (nano-DSF). Microscale thermophoresis (MST) and yeast surface display (YSD) were performed to measure the relative affinities between the cognate toxin-antitoxin partners. The interaction between MazEF6 complexes and cognate promoter DNA was also studied using MST. Analysis of paired-end RNA sequencing data revealed that the overexpression of MazF6 resulted in differential expression of 323 transcripts in M. tuberculosis. Network analysis was performed to identify the nodes from the top-response network. The analysis of mRNA protection ratios resulted in identification of putative MazF6 cleavage site in its native host, M. tuberculosis. IMPORTANCE M. tuberculosis harbors a large number of type II toxin-antitoxin (TA) systems, the exact roles for most of which are unclear. Prior studies have reported that overexpression of several of these type II toxins inhibits bacterial growth and contributes to the formation of drug-tolerant populations in vitro. To obtain insights into M. tuberculosis MazEF6 type II TA system function, we determined stability, oligomeric states, and binding affinities of cognate partners with each other and with their promoter operator DNA. Using RNA-seq data obtained from M. tuberculosis overexpression strains, we have identified putative MazF6 cleavage sites and targets in its native, cellular context.

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Biochemical Investigation of Rv3404c from Mycobacterium tuberculosis.

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Biochemical Investigation of Rv3404c from Mycobacterium tuberculosis.

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