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Adaptive metabolic reprogramming gives cancer cells a proliferative advantage. Tumour cells extensively use glycolysis to sustain anabolism and produce serine, which not only refuels the one-carbon units necessary for the synthesis of nucleotide precursors and for DNA methylation, but also affects the cellular redox homeostasis. Given its central role in serine metabolism, serine hydroxymethyltransferase ( SHMT), a pyridoxal 5′-phosphate ( PLP)-dependent enzyme, is an attractive target for tumour chemotherapy. In humans, the cytosolic isoform ( SHMT1) and the mitochondrial isoform ( SHMT2) have distinct cellular roles, but high sequence identity and comparable catalytic properties, which may complicate development of successful therapeutic strategies. Here, we investigated how binding of the cofactor PLP controls the oligomeric state of the human isoforms. The fact that eukaryotic SHMTs are tetrameric proteins while bacterial SHMTs function as dimers may suggest that the quaternary assembly in eukaryotes provides an advantage to fine-tune SHMT function and differentially regulate intertwined metabolic fluxes, and may provide a tool to address the specificity problem. We determined the crystal structure of SHMT2, and compared it to the apo-enzyme structure, showing that PLP binding triggers a disorder-to-order transition accompanied by a large rigid-body movement of the two cofactor-binding domains. Moreover, we demonstrated that SHMT1 exists in solution as a tetramer, both in the absence and presence of PLP, while SHMT2 undergoes a dimer-to-tetramer transition upon PLP binding. These findings indicate an unexpected structural difference between the two human SHMT isoforms, which opens new perspectives for understanding their differing behaviours, roles or regulation mechanisms in response to PLP availability in vivo. [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]

The intracellular level of the bacterial secondary messenger cyclic di-3',5'-GMP (c-di-GMP) is determined by a balance between its biosynthesis and degradation, the latter achieved via dedicated phosphodiesterases (PDEs) bearing a characteristic EAL or HD-GYP domain. We here report the crystal structure of PA4781, one of the three Pseudomonas aeruginosa HD-GYP proteins, which we have previously characterized in vitro. The structure shows a bimetallic active site whose metal binding mode is different from those of both HD-GYP PDEs characterized so far. Purified PA4781 does not contain iron in the active site as for other HD-GYPs, and we show that it binds to a wide range of transition metals with similar affinities. Moreover, the structural features of PA4781 indicate that this is preferentially a pGpG binding protein, as we previously suggested. Our results point out that the structural features of HD-GYPs are more complex than predicted so far and identify the HD-GYP domain as a conserved scaffold which has evolved to preferentially interact with a partner GGDEF but which harbors different functions obtained through diversification of the active site. [ABSTRACT FROM AUTHOR]