Calcium oxalate (CaOx) crystals are distributed among all taxonomic levels of photosynthetic organisms from small algae to angiosperms and giant gymnosperms. Accumulation of crystals by these organisms can be substantial. Major functions of CaOx crystal formation in plants include high-capacity calcium (Ca) regulation and protection against herbivory. Ultrastructural and developmental analyses have demonstrated that this biomineralization process is not a simple random physical-chemical precipitation of endogenously synthesized oxalic acid and environmentally derived Ca. Instead, crystals are formed in specific shapes and sizes. Genetic regulation of CaOx formation is indicated by constancy of crystal morphology within species, cell specialization, and the remarkable coordination of crystal growth and cell expansion. Using a variety of approaches, researchers have begun to unravel the exquisite control mechanisms exerted by cells specialized for CaOx formation that include the machinery for uptake and accumulation of Ca, oxalic acid biosynthetic pathways, and regulation of crystal growth.

CALCIUM OXALATE IN PLANTS: Formation and Function

Genes involved in fatty acid catabolism have undergone extensive duplication in the genus Mycobacterium, which includes the etiologic agents of leprosy and tuberculosis Here, we show that prokaryotic- and eukaryotic-like isoforms of the glyoxylate cycle enzyme isocitrate lyase (ICL) are jointly required for fatty acid catabolism and virulence in Mycobacterium tuberculosis Although deletion of icl1 or icl2, the genes that encode ICL1 and ICL2, respectively, had little effect on bacterial growth in macrophages and mice, deletion of both genes resulted in complete impairment of intracellular replication and rapid elimination from the lungs The feasibility of targeting ICL1 and ICL2 for chemical inhibition was shown using a dual-specific ICL inhibitor, which blocked growth of M tuberculosis on fatty acids and in macrophages The absence of ICL orthologs in mammals should facilitate the development of glyoxylate cycle inhibitors as new drugs for the treatment of tuberculosis

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Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence

Analysis by two-dimensional gel electrophoresis revealed that Mycobacterium avium expresses several proteins unique to an intracellular infection. One abundant protein with an apparent molecular mass of 50 kDa was isolated, and the N-terminal sequence was determined. It matches a sequence in the M. tuberculosis database (Sanger) with similarity to the enzyme isocitrate lyase of both Corynebacterium glutamicum and Rhodococcus fascians. Only marginal similarity was observed between this open reading frame (ORF) (termed icl) and a second distinct ORF (named aceA) which exhibits a low similarity to other isocitrate lyases. Both ORFs can be found as distinct genes in the various mycobacterial databases recently published. Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anapleurotic enzyme for growth on acetate and certain fatty acids as carbon source. In this study we express and purify Icl, as well as AceA proteins, and show that both exhibit isocitrate lyase activity. Various known inhibitors for isocitrate lyase were effective. Furthermore, we present evidence that in both M. avium and M. tuberculosis the production and activity of the isocitrate lyase is enhanced under minimal growth conditions when supplemented with acetate or palmitate.

Characterization of Activity and Expression of Isocitrate Lyase in Mycobacterium avium and Mycobacterium tuberculosis

An impediment to the rational development of novel drugs against tuberculosis (TB) is a general paucity of knowledge concerning the metabolism of Mycobacterium tuberculosis, particularly during infection. Constraint-based modeling provides a novel approach to investigating microbial metabolism but has not yet been applied to genome-scale modeling of M. tuberculosis. GSMN-TB, a genome-scale metabolic model of M. tuberculosis, was constructed, consisting of 849 unique reactions and 739 metabolites, and involving 726 genes. The model was calibrated by growing Mycobacterium bovis bacille Calmette Guerin in continuous culture and steady-state growth parameters were measured. Flux balance analysis was used to calculate substrate consumption rates, which were shown to correspond closely to experimentally determined values. Predictions of gene essentiality were also made by flux balance analysis simulation and were compared with global mutagenesis data for M. tuberculosis grown in vitro. A prediction accuracy of 78% was achieved. Known drug targets were predicted to be essential by the model. The model demonstrated a potential role for the enzyme isocitrate lyase during the slow growth of mycobacteria, and this hypothesis was experimentally verified. An interactive web-based version of the model is available. The GSMN-TB model successfully simulated many of the growth properties of M. tuberculosis. The model provides a means to examine the metabolic flexibility of bacteria and predict the phenotype of mutants, and it highlights previously unexplored features of M. tuberculosis metabolism.

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GSMN-TB: a web-based genome-scale network model of Mycobacterium tuberculosis metabolism

Mononuclear Iron Enzymes Are Primary Targets of Hydrogen Peroxide Stress

Article de synthese sur l'isocitrate lyase, isolement, comparaison des techniques de purification, etude de sa structure, de sa specificite, de sa stabilite et analyse cinetique

Comparative structure, function and regulation of isocitrate lyase, an important assimilatory enzyme.

Comparative biochemistry of the glyoxylate cycle

Isocitrate lyase from higher plants

We found that the Mg-isocitrate complex is the true substrate for pine isocitrate lyase and that magnesium acts as a non-essential activator. Both the non-activated and the activated enzyme forms are catalytically active. Our model is consistent with the presence of two Mg-binding sites with different affinities: an activator site with high affinity in addition to the catalytic site with lower affinity. This may result in a complex, fine regulation of isocitrate lyase activity by magnesium. The affinity of the free enzyme for isocitrate is very low. Moreover, free isocitrate does not bind to the activated enzyme, nor it can yield a catalytically active form by binding to an enzyme species whose catalytic site has already been bound by magnesium.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1988.tb13859.x

Giancarlo Pinzauti

Papers

Comparative structure, function and regulation of isocitrate lyase, an important assimilatory enzyme.

Comparative biochemistry of the glyoxylate cycle

Isocitrate lyase from higher plants

Isocitrate lyase from Pinus pinea: characterization of its true substrate and the action of magnesium ions

An isocitrate lyase of higher plants: Analysis and comparison of some molecular properties