MSACL 2017 US Abstract

A New Pathway for Glutaminolysis in Mycobacterium tuberculosis

Robert Jansen (Presenter)
Weill Cornell Medicine

Bio: Robert Jansen obtained his PhD from Utrecht University where he developed quantitative LC-MS/MS methods for the detection of therapeutic nucleotide analogs in white blood cells (advisor: Jos Beijnen). During postdoctoral research at the Netherlands Cancer Institute he applied untargeted LC-MS metabolomics to discover the endogenous substrates and function of human ABC-transporters (advisor: Piet Borst). Currently at Weill Cornell Medicine, he applies untargeted LC-MS metabolomics to discover the function of essential proteins of unknown function in Mycobacterium tuberculosis (advisor: Kyu Rhee).

Authorship: Robert Jansen (1), Jessica Pinkham (2), Xuelin Bian (3), James Sacchettini (3), Eric Rubin (2), Kyu Rhee (1)
(1) Weill Cornell Medicince, New York, NY, (2) Harvard T.H. Chan School of Public Health, Boston, MA, (3) Texas A&M University, College Station, TX

Short Abstract

Mycobacterium tuberculosis (MTB) is the leading cause of deaths due to an infectious agent, killing 1.5 million people in 2014. Genetic screens have shown that the gene Rv3722c is essential for MTB, but its function is unknown. Using activity-based metabolite profiling and untargeted metabolomics, we show that Rv3722c is an aminotransferase that converts glutamine into its keto acid oxoglutaramate. Comparative genomics revealed that another gene of unknown function, Rv0480c, functions as an omega-amidase that hydrolyzes oxoglutaramate to alpha-ketoglutarate and free ammonia. Together, Rv3722c and Rv0480c constitute a non-canonical pathway for glutaminolysis that affects the carbon-nitrogen balance in MTB.

Long Abstract

Mycobacterium tuberculosis (MTB) killed 1.5 million people in 2014, making it the leading cause of deaths due to an infectious agent. Genetic screens have shown that the gene Rv3722c is essential for MTB, but its function is unknown.

Incubation of purified recombinant Rv3722c with a small metabolite library revealed time- and Rv3722c-dependent formation of a metabolite with an m/z of 144.030 [M-H]-. Using an in-house synthesized standard, this metabolite could be identified as oxoglutaramate, the keto acid of glutamine. Enzyme characterization with pure substrates showed that Rv3722c is an aminotransferase with a broad substrate-specificity in-vitro. Untargeted metabolomics analysis of an Rv3722c-deficient MTB strain revealed extensive accumulation of glutamine, indicating that glutamine is a preferred substrate in vivo.

In other organisms oxoglutaramate is hydrolyzed to alpha-ketoglutarate and free ammonia by omega-amidases. Comparative genomics suggested that Rv0480c, another gene of unknown function, could function as an omega-amidase that hydrolyzes oxoglutaramate in MTB. Purified recombinant Rv0480c indeed catalyzed the hydrolysis of oxoglutaramate.

Together, Rv3722c and Rv0480c constitute a non-canonical pathway for glutaminolysis. Interestingly, stable isotope tracing after incubation with 15N-amide and 15N-amine-labeled glutamine showed that the canonical glutaminolysis through glutamine oxoglutarate aminotransferase (GOGAT) is not the main pathway used by MTB under standard culture conditions. We are currently investigating the function of the Rv3722c-Rv0480c pathway.


References & Acknowledgements:

This work was supported in part by the NIH grant U19 AI107774.


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