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De novo thymidylate synthesis is a crucial pathway for normal and cancer cells. Deoxythymidine monophosphate (dTMP) is synthesized by the combined action of three enzymes: serine hydroxymethyltransferase (SHMT1), dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS), with the latter two being targets of widely used chemotherapeutics such as antifolates and 5‐fluorouracil. These proteins translocate to the nucleus after SUMOylation and are suggested to assemble in this compartment into the thymidylate synthesis complex. We report the intracellular dynamics of the complex in cancer cells by an in situ proximity ligation assay, showing that it is also detected in the cytoplasm. This result indicates that the role of the thymidylate synthesis complex assembly may go beyond dTMP synthesis. We have successfully assembled the dTMP synthesis complex in vitro, employing tetrameric SHMT1 and a bifunctional chimeric enzyme comprising human thymidylate synthase and dihydrofolate reductase. We show that the SHMT1 tetrameric state is required for efficient complex assembly, indicating that this aggregation state is evolutionarily selected in eukaryotes to optimize protein–protein interactions. Lastly, our results regarding the activity of the complete thymidylate cycle in vitro may provide a useful tool with respect to developing drugs targeting the entire complex instead of the individual components. [ABSTRACT FROM AUTHOR]

Bis‐(3′‑5′)‐cyclic diguanylic acid (c‑di‑GMP) belongs to the class of cyclic dinucleotides, key carriers of cellular information in prokaryotic and eukaryotic signal transduction pathways. In bacteria, the intracellular levels of c‐di‐GMP and their complex physiological outputs are dynamically regulated by environmental and internal stimuli, which control the antagonistic activities of diguanylate cyclases (DGCs) and c‐di‐GMP specific phosphodiesterases (PDEs). Allostery is one of the major modulators of the c‐di‐GMP‐dependent response. Both the c‐di‐GMP molecule and the proteins interacting with this second messenger are characterized by an extraordinary structural plasticity, which has to be taken into account when defining and possibly predicting c‐di‐GMP‐related processes. Here, we report a structure‐function relationship study on the catalytic portion of the PA0575 protein from Pseudomonas aeruginosa, bearing both putative DGC and PDE domains. The kinetic and structural studies indicate that the GGDEF‐EAL portion is a GTP‐dependent PDE. Moreover, the crystal structure confirms the high degree of conformational flexibility of this module. We combined structural analysis and protein engineering studies to propose the possible molecular mechanism guiding the nucleotide‐dependent allosteric control of catalysis; we propose that the role exerted by GTP via the GGDEF domain is to allow the two EAL domains to form a dimer, the species competent to enter PDE catalysis. The catalytic portion (GGDEF‐EAL domains in tandem) of the Pseudomonas aeruginosa PA0575 protein (RmcA) is a phosphodiesterase (PDE) allosterically controlled by GTP. The crystal structure confirms the large plasticity of this motif; we propose that this feature allows the two EAL domains to form a dimer, the catalytically competent species, upon GTP binding to the GGDEF module. [ABSTRACT FROM AUTHOR]

Cancer cells reprogramme one‐carbon metabolism (OCM) to sustain growth and proliferation. Depending on cell demands, serine hydroxymethyltransferase (SHMT) dynamically changes the fluxes of OCM by reversibly converting serine and tetrahydrofolate (THF) into 5,10‐methylene‐THF and glycine. SHMT is a tetrameric enzyme that mainly exists in three isoforms; two localize in the cytosol (SHMT1/SHMT2α) and one (SHMT2) in the mitochondria. Both the cytosolic isoforms can also translocate to the nucleus to sustain de novo thymidylate synthesis and support cell proliferation. Finally, the expression levels of the different isoforms are regulated to a certain extent by a yet unknown crosstalk mechanism. We have designed and fully characterized a set of three SHMT1 mutants, which uncouple the oligomeric state of the enzyme from its catalytic activity. We have then investigated the effects of the mutations on SHMT1 nuclear localization, cell viability and crosstalk in lung cancer cells (A549; H1299). Our data reveal that in these cell lines de novo thymidylate synthesis requires SHMT1 to be active, regardless of its oligomeric state. We have also confirmed that the crosstalk between the cytosolic and mitochondrial SHMT actually takes place and regulates the expression of the two isoforms. Apparently, the crosstalk mechanism is independent from the oligomeric state and the catalytic activity of SHMT1. Database: Structural data are available in the PDB under the accession number 6FL5 [ABSTRACT FROM AUTHOR]

In the denitrification pathway, Pseudomonas aeruginosa cytochrome cd1 nitrite reductase catalyzes the reduction of nitrite to nitric oxide; in vitro, this enzyme is also competent in the reduction of O2 to 2H2O. In this article, we present a comparative kinetic study of the O2 reaction in the wild-type nitrite reductase and in three site-directed mutants (Tyr10-->Phe, His369-->Ala and His327-->Ala/His369-->Ala) of the amino acid residues close to the d1 heme on the distal side. The results clearly indicate that His369 is the key residue in the control of reactivity, as its substitution with Ala, previously shown to affect the reduction of nitrite, also impairs the reaction with O2, affecting both the properties and lifespan of the intermediate species. Our findings allow the presentation of an overall picture for the reactivity of cytochrome cd1 nitrite reductase and extend our previous conclusion that the conserved distal histidines are essential for the binding to reduced d1 heme of different anions, whether a substrate such as nitrite, a ligand such as cyanide, or an intermediate in the O2 reduction. Moreover, we propose that His369 also exerts a protective role against degradation of the d1 heme, by preventing the formation and adverse effects of the reactive O2 species (never present in significant amounts in wild-type cytochrome cd1 nitrite reductase), a finding with clear physiological implications.