O2-independent hydroxylation and the anaerobic biosynthesis of ubiquinone – O2-taboo

Task 1 of the project aims at understanding the control and the physiology of the O2-independent UQ pathway not only under anaerobiosis, but also under microaerobiosis (low concentrations of O2). For this, we’ll study the importance and the regulation of the pathway by constructing Escherichia coli mutants for the diverse quinone biosynthetic pathways and we’ll study the phenotypes of the strains under atmospheres with controlled O2 levels, and also under physiological conditions in the mouse intestine. Task 2 aims at understanding how UbiU and UbiV function. These two proteins represent a new class of enzymes which catalyze hydroxylation reactions independently from O2. To do so, we’ll employ various techniques like organic chemistry (for the synthesis of substrates and labeled molecules), biochemistry (to purify protein and for spectroscopic and enzymatic studies), structural biology (to solve the 3D structures of proteins by X ray crystallography). Task 3 ambitions to establish the role of the UbiT protein and to verify if the Ubi proteins of the O2-independent pathway form a multiprotein complex, similar to what we showed for the O2-dependent pathway. In order to reveal and characterize the putative complex, we’ll combine biochemistry, structural biology and synthetic biology. These approaches will be guided by bioinformatic studies of amino acid coevolution to predict interaction surfaces between Ubi proteins. Finally, the fourth task of the project will employ genomics and phylogenetic approaches in order to (i) precisely document which quinone pathways are present in the wide range of bacterial species, (ii) define which pathway (O2-dependent or O2-independent) evolved first, likely around 2.4 billion years ago.

Task1 : The genetic analysis of the O2-independent UQ pathway revealed the implication of the aro/tyr pathway and pointed to a probable role of prephenate as an oxygen donor for the hydroxylation reactions catalyzed by UbiU-V. The first results suggest that E. coli strains mutated for ubiU-V show a defect during the early phase of the colonization of the intestine of mice.
Task 2 : Prephenate being unstable, partner 2 (P2) purified a domain of the PheA protein which allows the in situ production of prephenate from commercially available chorismate. This will facilitate the development of the in vitro activity assay for UbiU-V for which substrates analogs with short chains have already been synthesized (farnesyl-phenol and farnesyl-catechol). P2 also started crystallization trials with the purified complex UbiU-V. A genetic approach undertaken by P3 showed that UbiU-V are able to function in the presence of O2, which is an interesting result given the presence of 4Fe-4S clusters (usually sensitive to O2) in both proteins.
Task 3 : P1 demonstrated that the UbiT protein is part of the Ubi complex which gathers most proteins involved in the O2-dependent production of ubiquinone. P1 also started to construct a library of plasmids expressing the 8 proteins of the Ubi complex in order to overproduce it and allow structural studies.
Task 4 : P1 finalized a bioinformatic tool for the automatic annotation of the ubiquinone and menaquinone pathways in bacterial genomes. This will allow the analysis of the large scale distribution of these pathways and is a pre-requisite for the study of the evolutionary history of the ubiquinone pathways.

The good progress made so far support that the initial objectives should be completed.

P1, P2, P3 demonstrated that the 3 genes specific to the O2-independent pathway (ubiT-V) are essential to the denitrification process in the pathogenic bacterium Pseudomonas aeruginosa and that the UbiT protein is capable of binding the polyprenyl chain of the biosynthetic intermediates of UQ. These results, published in JBC in may 2020 (doi : 10.1074/jbc.RA120.013748), establish the physiological importance of the O2-independent pathway and characterize a chaperone role for UbiT.
A review about the complexity and diversity of the UQ biosynthetic pathways in bacteria has been published by P1 in BBA - Bioenergetics in 2020 (doi : 10.1016/j.bbabio.2020.148259).

Isoprenoid quinones are central for cellular physiology since they act as electrons and protons shuttles in energy-generating respiratory chains and in various processes like haem and uracil biosynthesis or disulfide bond formation. Escherichia coli and many proteobacteria possess two types of isoprenoid quinones: ubiquinone (UQ) and (demethyl)menaquinone (D)MK. Typically, UQ is considered as an “aerobic” quinone since it participates to aerobic respiration and its biosynthesis requires dioxygen (O2) for three hydroxylation steps. In contrast (D)MK have been characterized as “anaerobic” quinones as they participate to anaerobic respiration and their synthesis doesn’t depend on O2.
During the AnaeroUbi project supported by ANR (2015-2019), our consortium has discovered that E. coli can synthesize UQ by an O2-independent pathway. We identified three genes ubiT, ubiU and ubiV that are essential for the biosynthesis of UQ in anaerobic conditions . Furthermore, we found that the O2-independent UQ pathway is widely conserved in proteobacteria. Thus, our results revisit the current dogma about UQ being an aerobic quinone and show that bacteria actually synthesize UQ over the entire range of O2 concentrations. Our multidisciplinary O2-taboo project is based on this discovery and will address important issues in four interconnected tasks:

1) We propose to evaluate the regulation of the O2-independent UQ pathway and its contribution to bacterial physiology. Experiments will be conducted in laboratory cultures at various O2 levels and also inside a natural host of E. coli, where low O2 conditions prevail. Our results are likely to be applicable, at least in part, to most facultative anaerobic bacteria that count many human pathogens.
2) Three O2-independent hydroxylation steps are required in the newly-discovered UQ pathway. We have accumulated preliminary evidence that UbiU and UbiV represent a novel class of O2-independent hydroxylases. We propose to elucidate their structure, their catalytic mechanism, the role of their Fe-S clusters and to establish the identity of the OH donor in these hydroxylation reactions.
3) In the cell, the biosynthesis of UQ demands that highly hydrophobic substrates are sequentially modified by multiple enzymes. We showed recently that the O2-dependent biosynthesis of UQ takes place inside a multiprotein complex, in which UbiJ binds the hydrophobic biosynthetic intermediates via its SCP2 domain. Our preliminary data support that the O2-independent pathway organizes in a complex around the SCP2-containing UbiT protein. We propose to characterize the O2-independent complex by identifying its components and their interactions, using a combination of biochemical and bioinformatic approaches.
4) Finally, we propose to study the evolution of the O2-independent pathway to evaluate if it appeared before or after the O2-dependent pathway. Our results could challenge the current paradigm that consists in the requirement for high O2 concentration habitats for the appearance of the UQ pathway on Earth, about 2.4 billion years ago.

By combining complementary approaches, the O2-taboo project addresses forefront questions in microbial physiology, cellular biochemistry and evolutionary biology. Overall, our results will contribute significant advances by elucidating (i) the regulation and physiological importance of a new pathway conserved in proteobacteria, (ii) the molecular function and supramolecular organization of the UbiT,U,V proteins, (iii) the unprecedented chemistry of novel O2-independent hydroxylation reactions, (iv) the distribution and evolution of the UQ pathways in proteobacteria.