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Human serine racemase (hSR) catalyzes the biosynthesis of D‐serine, an obligatory co‐agonist of the NMDA receptors. It was previously found that the reversible S‐nitrosylation of Cys113 reduces hSR activity. Here, we show by site‐directed mutagenesis, fluorescence spectroscopy, mass spectrometry, and molecular dynamics that S‐nitrosylation stabilizes an open, less‐active conformation of the enzyme. The reaction of hSR with either NO or nitroso donors is conformation−dependent and occurs only in the conformation stabilized by the allosteric effector ATP, in which the ε‐amino group of Lys114 acts as a base toward the thiol group of Cys113. In the closed conformation stabilized by glycine—an active‐site ligand of hSR—the side chain of Lys114 moves away from that of Cys113, while the carboxyl side‐chain group of Asp318 moves significantly closer, increasing the thiol pKa and preventing the reaction. We conclude that ATP binding, glycine binding, and S‐nitrosylation constitute a three‐way regulation mechanism for the tight control of hSR activity. We also show that Cys113 undergoes H2O2‐mediated oxidation, with loss of enzyme activity, a reaction also dependent on hSR conformation. [ABSTRACT FROM AUTHOR]

The exploitation of methionine-depleting enzyme methionine γ-lyase (MGL) is a promising strategy against specific cancer cells that are strongly dependent on methionine. To identify MGL from different sources with high catalytic activity and efficient anticancer action, we have expressed and characterized MGL from Clostridium novyi and compared its catalytic efficiency with the previously studied MGL from Citrobacter freundii. The purified recombinant MGL exhibits kcat and kcat/Km for methionine γ-elimination reaction that are 2.4- and 1.36-fold higher than C. freundii enzyme, respectively, whereas absorption, fluorescence, and circular dichroism spectra are very similar, as expected on the basis of 87% sequence identity and high conservation of active site residues. The reactivity of cysteine residues with DTNB and iodoacetamide was investigated as well as the impact of their chemical modification on catalytic activity. This information is relevant because for increasing bioavailability and reducing immunogenity, MGL should be decorated with polyethylene glycol (PEG). It was found that Cys118 is a faster reacting residue, which results in a significant decrease in the γ-elimination activity. Thus, the protection of Cys118 before conjugation with cysteine-reacting PEG represents a valuable strategy to preserve MGL activity. The anticancer action of C. novyi MGL, evaluated in vitro against prostate (PC-3), chronic myelogenous leucemia (K562), and breast (MDA-MB-231 and MCF7) cancer cells, exhibits IC50 of 1.3 U mL−1, 4.4 U mL−1, 1.2 U mL−1, and 3.4 U mL−1, respectively. A higher cytotoxicity of C. novyi MGL was found against cancer cells with respect to C. freundii MGL, with the exception of PC-3, where a lower cytotoxicity was observed. © 2017 IUBMB Life, 69(9):668-676, 2017 [ABSTRACT FROM AUTHOR]

Abstract: Protein ubiquitination occurs through formation of an isopeptide bond between the C-terminal glycine of ubiquitin (Ub) and the ɛ-amino group of a substrate lysine residue. This post-translational modification, which occurs through the attachment of single and/or multiple copies of mono-ubiquitin and poly-ubiquitin chains, is involved in crucial cellular events such as protein degradation, cell-cycle regulation and DNA repair. The abnormal functioning of ubiquitin pathways is also implicated in the pathogenesis of several human diseases ranging from cancer to neurodegeneration. However, despite the undoubted biological importance, understanding the molecular basis of how ubiquitination regulates different pathways has up to now been strongly limited by the difficulty of producing the amounts of highly homogeneous samples that are needed for a structural characterization by X-ray crystallography and/or NMR. Here, we report on the production of milligrams of highly pure Josephin mono-ubiquitinated on lysine 117 through large scale in vitro enzymatic ubiquitination. Josephin is the catalytic domain of ataxin-3, a protein responsible for spinocerebellar ataxia type 3. Ataxin-3 is the first deubiquitinating enzyme (DUB) reported to be activated by mono-ubiquitination. We demonstrate that the samples produced with the described method are correctly folded and suitable for structural studies. The protocol allows facile selective labelling of the components. Our results provide an important proof-of-concept that may pave the way to new approaches to the in vitro study of ubiquitinated proteins. [Copyright &y& Elsevier]

Type 1 nonsymbiotic hemoglobin from Arabidopsis thaliana (AHb1) shows a partial bis-histidyl hexacoordination but can reversibly bind diatomic ligands. The physiological function is still unclear, but the high oxygen affinity rules out a function related to O2 sensing, carrying, or storing. To gain insight into its possible functional roles, we have investigated its O2 and NO rebinding kinetics after laser flash photolysis. The rate constants of the rebinding from the primary docking site for both O2 and NO are higher than CO, with lower photolysis yields. Moreover, the amplitude of the geminate phase increases and, as for CO, the numerical analysis of the experimental curves suggests the existence of an internal pathway leading, with high rate, to an additional docking site. However, the accessibility to this site seems to be strongly ligand-dependent, being lower for O2 and higher for NO. © 2011 IUBMB IUBMB Life, 63(12): 1094-1100, 2011 [ABSTRACT FROM AUTHOR]