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Toxoplasmosis persists as a prevalent disease, facing challenges from parasite resistance and treatment side effects. Consequently, identifying new drugs by exploring novel protein targets is essential for effective intervention. Cyclosporin A (CsA) possesses antiparasitic activity against Toxoplasma gondii, with cyclophilins identified as possible targets. However, CsA immunosuppressive nature hinders its use as an antitoxoplasmosis agent. Here, we evaluate the potential of three CsA derivatives devoid of immunosuppressive activity, namely, NIM811, Alisporivir, and dihydrocyclosporin A to target a previously characterized cyclophilin from Toxoplasma gondii (TgCyp23). We determined the X‐ray crystal structures of TgCyp23 in complex with the three analogs and elucidated their binding and inhibitory properties. The high resolution of the structures revealed the precise positioning of ligands within the TgCyp23 binding site and the details of protein–ligand interactions. A comparison with the established ternary structure involving calcineurin indicates that substitutions at position 4 in CsA derivatives prevent calcineurin binding. This finding provides a molecular explanation for why CsA analogs can target Toxoplasma cyclophilins without compromising the human immune response. [ABSTRACT FROM AUTHOR]

Homocystinuria is a rare disease caused by mutations in the CBS gene that results in a deficiency of cystathionine γ-synthase (CBS). CBS is an essential pyridoxal 50-phosphate (PLP)-dependent enzyme in the transsulfuration pathway, responsible for combining serine with homocysteine to produce cystathionine, whose activity is enhanced by the allosteric regulator S-adenosylmethionine (SAM). CBS also plays a role in generating hydrogen sulfide (H2S), a gaseous signaling molecule with diverse regulatory functions within the vascular, nervous, and immune systems. In this study, we present the clinical and biochemical characterization of two novel CBS missense mutations that do not respond to pyridoxine treatment, namely c.689T > A (L230Q) and 215A > T (K72I), identified in a Chinese patient. We observed that the disease-associated K72I genetic variant had no apparent effects on the spectroscopic and catalytic properties of the full-length enzyme. In contrast, the L230Q variant expressed in Escherichia coli did not fully retain heme and when compared with the wild-type enzyme, it exhibited more significant impairments in both the canonical cystathionine-synthesis and the alternative H2S-producing reactions. This reduced activity is consistent with both in vitro and in silico evidence, which indicates that the L230Q mutation significantly decreases the overall protein's stability, which in turn, may represent the underlying cause of its pathogenicity. [ABSTRACT FROM AUTHOR]

Carbon monoxide (O) migration and binding in the nonsymbiotic hemoglobins AHb1 and AHb2 of Arabidopsis thaliana is studied by using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures. It is shown that geminate rebinding properties are different for the two conformations and docking site C is not accessible in the low-pH conformation.

Pyridoxal 5′-phosphate (PLP)-dependent enzymes catalyze a wide range of reactions of amino acids and amines, with the exception of glycogen phosphorylase which exhibits peculiar both substrate preference and chemical mechanism. They represent about 4% of the gene products in eukaryotic cells. Although structure-function investigations regarding these enzymes are copious, their regulation by post-translational modifications is largely unknown. Protein phosphorylation is the most common post-translational modification fundamental in mediating diverse cellular functions. This review aims at summarizing the current knowledge on regulation of PLP enzymes by phosphorylation. Starting from the paradigmatic PLP-dependent glycogen phosphorylase, the first phosphoprotein discovered, we collect data in literature regarding functional phosphorylation events of eleven PLP enzymes belonging to different fold types and discuss the impact of the modification in affecting their activity and localization as well as the implications on the pathogenesis of diseases in which many of these enzymes are involved. The pivotal question is to correlate the structural consequences of phosphorylation among PLP enzymes of different folds with the functional modifications exerted in terms of activity or conformational changes or others. Although the literature shows that the phosphorylation of PLP enzymes plays important roles in mediating diverse cellular functions, our recapitulation of clue findings in the field makes clear that there is still much to be learnt. Besides mass spectrometry-based proteomic analyses, further biochemical and structural studies on purified native proteins are imperative to fully understand and predict how phosphorylation regulates PLP enzymes and to find the relationship between addition of a phosphate moiety and physiological response. [ABSTRACT FROM AUTHOR]