Despite a general belief according to which central metabolism, especially in prokaryotes, has nothing left to reveal, our results suggest that certain bacteria like M. tuberculosis, the agent of tuberculosis, could possess an hybrid ‘supercomplex’ which could merge two key metabolic reactions. Our goal is to capture and study this complex that has no known analogue in other species.
The primary objective of this project is first to validate the presence, in Actinobacteria, of an hybrid supercomplex between the tripartite complexes of PDH (pyruvate dehydrogenase) and KDH (alpha-ketoglutarate dehydrogenase), and then to purify it to shed light on its structure and its regulation. In addition to study how such a huge complex, which might be larger than the ribosome, could be structured and coordinate the different biochemical reactions that take place inside it, our purpose and challenge is to understand which kind of advantages an hybrid complex could provide to central metabolism, especially in the case of M. tuberculosis. Little is known about how this pathogen adapts inside the human host where it may persist indefinitely into a dormant state.
Our objectives will be achieved mostly by a biochemical approach:
- first to either purify the complex from total cell extracts of Corynebacterium glutamicum and/or Mycobacterium smegmatis, both non pathogenic Actinobacteria, or to reconstitute the complex in vitro from its four component proteins produced in Escherichia coli;
- to characterize the assembly (protein-protein interactions and affinities) and the architecture of the complex by an integrative structural biology approach including X-ray crystallography of the components, small-angle X-ray scattering (SAXS), analytical ultracentrifugation, cryo-EM, molecular modelling;
- to study the structure and the dynamics of the complex, focalizing on regulation aspects.
The main results achieved so far (June 2015) can be resumed as follows:
i. Crystal structures of the catalytic domain of C. glutamicum E2p (dihydrolipoamide acetyltransferase), the component predicted to form the complex core, in the apo form as well as in complex with CoA-SH, the acceptor substrate, or the product acetyl-CoA;
ii. Crystal structure of C. glutamicum E1p (pyruvate dehydrogenase);
iii. Analysis of the oligomerisation and the solution state of E2p, either whole protein or the C-terminal catalytic domain, by analytical ultracentrifugation and SAXS. This experiment confirm the homotrimeric base state of E2, also for the whole protein, and show – as expected – a quite extended conformation, consistently with the presence of the additional ‘lipoyl’ and peripheral-subunit binding domains, which do interact with the other complex components.
This project primarily seeks a 3D model of a huge metabolic complex, which presence in Actinobacteria is suggested by experimental evidence but whose structure is unknown – a technical challenge in itself. However, our long-term goal is to go beyond a static picture and to understand how such a complex could be regulated. This may open exciting perspectives for the development of new antibiotics, also keeping in mind that new control mechanisms of the bacterial central metabolism, not necessarily restricted to Actinobacteria, could be unveiled.
Tuberculosis remains nowadays a major threat for public health, with up to one third of the world population estimated to be latently infected with Mycobacterium tuberculosis and therefore susceptible to develop active disease at anytime through reactivation, especially in case of weakening of the immune response. To become able to target this huge reservoir in an effective way would constitute undoubtedly a great progress towards the eradication of the disease. Indeed, one of the biggest challenges in drug development against tuberculosis is how to hit dormant bacteria, a particularly difficult task in light of the fact that the molecular mechanisms underlying the adaptation of the pathogen to the different environmental conditions during the disease stages, notably dormancy, are still poorly understood. For these reasons we have been actively involved in the study of how M. tuberculosis controls its central metabolism, a critical process in order to switch between active growth and dormancy. Our recent work in this field has been focused on the structural characterization of a-ketoglutarate decarboxylase (KGD), a central enzyme in the tricarboxylic acid cycle (TCA) and part of the tripartite a-ketoglutarate dehydrogenase complex (KDH). First identified as an enzyme inhibited by GarA, an FHA-domain protein acting in turn as a sort of molecular switch controlled by phosphorylation, we showed that KGD is an extremely dynamic object able to carry out different reactions depending on the acyl acceptor substrate, and that its inhibition by GarA takes place through an unprecedented allosteric-like mechanism, by which the enzyme is frozen into a catalytically incompetent state.
Although the KDH complex was initially reported to be absent in M. tuberculosis due to failure to detect its activity, we now know that not only this activity can be measured under appropriate conditions but also that this complex may in reality form a unique supercomplex with pyruvate dehydrogenase (PDH), a similarly structured complex involved in the oxidative decarboxylation of pyruvate produced by glycolysis. Merging the two complexes into a hybrid one would generate a MDa-range multienzyme assembly that could span a diameter larger than the ribosome and without analogies in other organisms. The objective of this project is first to validate the existence of this supercomplex as a physiologically relevant enzymatic machinery in the Corynebacterineae suborder (that includes mycobacteria and corynebacteria), then to purify and characterize it by a combination of hybrid structural biology approaches to get a three-dimensional model of this supramolecular biological object. The ultimate goal, however, is to go beyond a static structural picture, clarifying the dynamic processes by which the different enzymatic activities may be temporally and spatially coordinated, and to understand, in the end, by which molecular mechanisms (and in response to which stimuli) such a huge machinery could be regulated.
Monsieur Marco Bellinzoni (Institut Pasteur) – firstname.lastname@example.org
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
IP Institut Pasteur
Help of the ANR 196,040 euros
Beginning and duration of the scientific project: December 2013 - 42 Months