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.
De novo thymidylate synthesis is a crucial pathway for cancer cells that rely on deoxythymidine monophosphate (dTMP) to fuel DNA synthesis. dTMP is synthesized by three enzymes forming a transient protein–protein complex. We report the intracellular dynamics and localization of the dTMP synthesis complex in cancer cells, as well as its successful assembly in vitro. Our results provide a useful tool to develop drugs targeting the entire complex instead of the individual components.
Nitrite, previously considered physiologically irrelevant and a simple end product of endogenous nitric oxide (NO) metabolism, is now envisaged as a reservoir of NO to be activated in response to ...oxygen (O(2)) depletion. In the first part of this review, we summarize and compare the mechanisms of nitrite-dependent production of NO in selected bacteria and in eukaryotes. Bacterial nitrite reductases, which are copper or heme-containing enzymes, play an important role in the adaptation of pathogens to O(2) limitation and enable microrganisms to survive in the human body. In mammals, reduction of nitrite to NO under hypoxic conditions is carried out in tissues and blood by an array of metalloproteins, including heme-containing proteins and molybdenum enzymes. In humans, tissues play a more important role in nitrite reduction, not only because most tissues produce more NO than blood, but also because deoxyhemoglobin efficiently scavenges NO in blood. In the second part of the review, we outline the significance of nitrite in human health and disease and describe the recent advances and pitfalls of nitrite-based therapy, with special attention to its application in cardiovascular disorders, inflammation, and anti-bacterial defence. It can be concluded that nitrite (as well as nitrate-rich diet for long-term applications) may hold promise as therapeutic agent in vascular dysfunction and ischemic injury, as well as an effective compound able to promote angiogenesis.
Aspergillus fumigatus is a saprophytic ubiquitous fungus whose spores can trigger reactions such as allergic bronchopulmonary aspergillosis or the fatal invasive pulmonary aspergillosis. To survive ...in the lungs, the fungus must adapt to a hypoxic and nutritionally restrictive environment, exploiting the limited availability of aromatic amino acids (AAAs) in the best possible way, as mammals do not synthesize them. A key enzyme for AAAs catabolism in A. fumigatus is AroH, a pyridoxal 5′‐phosphate‐dependent aromatic aminotransferase. AroH was recently shown to display a broad substrate specificity, accepting L‐kynurenine and α‐aminoadipate as amino donors besides AAAs. Given its pivotal role in the adaptability of the fungus to nutrient conditions, AroH represents a potential target for the development of innovative therapies against A. fumigatus‐related diseases. We have solved the crystal structure of Af‐AroH at 2.4 Å resolution and gained new insight into the dynamics of the enzyme's active site, which appears to be crucial for the design of inhibitors. The conformational plasticity of the active site pocket is probably linked to the wide substrate specificity of AroH.
•C heme nitrosylation is enhanced by nitrite reduction catalysis.•We analyze the nitrosylation of the c heme of the semiApo NiR.•We analyze the nitrosylation of both the c and the d1 hemes of the ...holo cd1NiR.
The reduction of nitrite into nitric oxide (NO) in denitrifying bacteria is catalyzed by nitrite reductase. In several species, this enzyme is a heme-containing protein with one c heme and one d1 heme per monomer (cd1NiR), encoded by the nirS gene.
For many years, the evidence of a link between NO and this hemeprotein represented a paradox, given that NO was known to tightly bind and, possibly, inhibit hemeproteins, including cd1NiRs.
It is now established that, during catalysis, cd1NiRs diverge from “canonical” hemeproteins, since the product NO rapidly dissociates from the ferrous d1 heme, which, in turn, displays a peculiar “low” affinity for NO (KD=0.11μM at pH 7.0).
It has been also previously shown that the c heme reacts with NO at acidic pH but c heme nitrosylation was not extensively investigated, given that in cd1NiR it was considered a side reaction, rather than a genuine process controlling catalysis.
The spectroscopic study of the reaction of cd1NiR and its semi-apo derivative (containing the sole c heme) with NO reported here shows that c heme nitrosylation is enhanced during catalysis; this evidence has been discussed in order to assess the potential of c heme nitrosylation as a regulatory process, as observed for cytochrome c nitrosylation in mammalian mitochondria.
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.
RNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of which have been characterized on ...structural grounds. Here, we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We have determined the cryoelectron microscopy (cryo-EM) structure of human SHMT1 in its free- and RNA-bound states, and we show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzyme’s reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in evolution of eukaryotic SHMT1 and in compartmentalization of one-carbon metabolism. Our findings provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.
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•Cryo-EM structure of human SHMT1 at 3.29 Å reveals different active site conformations•Cryo-EM structure of the SHMT1:RNA complex at 3.52 Å reveals a 4:1 binding stoichiometry•The tetrameric assembly and a flap structural motif are necessary for RNA binding•RNA riboregulates SHMT1 by competing with folate and acting as an allosteric switch
Using cryo-EM, Spizzichino et al. have solved the structure of the metabolic enzyme SHMT1 in the unbound form and in complex with RNA. Functional results showed that RNA riboregulates SHMT1 activity by acting as an allosteric switch, paving the way for the design of innovative RNA-based inhibitors.
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
Enzymes of intermediary metabolism are often reported to have moonlighting functions as RNA-binding proteins and have regulatory roles beyond their primary activities. Human serine ...hydroxymethyltransferase (SHMT) is essential for the one-carbon metabolism, which sustains growth and proliferation in normal and tumour cells. Here, we characterize the RNA-binding function of cytosolic SHMT (SHMT1) in vitro and using cancer cell models. We show that SHMT1 controls the expression of its mitochondrial counterpart (SHMT2) by binding to the 5′untranslated region of the SHMT2 transcript (UTR2). Importantly, binding to RNA is modulated by metabolites in vitro and the formation of the SHMT1–UTR2 complex inhibits the serine cleavage activity of the SHMT1, without affecting the reverse reaction. Transfection of UTR2 in cancer cells controls SHMT1 activity and reduces cell viability. We propose a novel mechanism of SHMT regulation, which interconnects RNA and metabolites levels to control the cross-talk between cytosolic and mitochondrial compartments of serine metabolism.
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