YbdK is a carboxylate-amine ligase with a γ-glutamyl:Cysteine ligase activity: Crystal structure and enzymatic assays (original) (raw)
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Interaction between the catalytic and modifier subunits of glutamate-cysteine ligase
Biochemical Pharmacology, 2007
b i o c h e m i c a l p h a r m a c o l o g y 7 4 ( 2 0 0 7 ) 3 7 2 -3 8 1 Glutamate-cysteine ligase Protein-protein interactions Yeast two-hybrid system Histidine-tagged protein Nickel-NTA column Oxidative stress JEL classification: Protein interactions a b s t r a c t Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the glutathione (GSH)
Mechanisms of γ-glutamylcysteine ligase regulation
Biochimica et Biophysica Acta (BBA) - General Subjects, 2006
The principal objective of this study was to investigate the mechanisms regulating the activity of γglutamylcysteine ligase (GCL; EC 6.3.2.2), the rate limiting enzyme in glutathione biosynthesis. Two phylogenetically divergent species, mouse and the fruitfly, Drosophila melanogaster were used to test the hypothesis that reversible protein phosphorylation and pyridine dinucleotide phosphate dependent allostery regulate GCL activity. GCL was almost completely inhibited under phosphorylating conditions, involving preincubations with MgATP and endogenous protein kinases. Maximal GCL inhibitions of 94%, 77%, 85%, 87%, 83%, 95% and 89% occurred, respectively, in mouse cerebellum, hippocampus, brainstem, striatum, cortex and heart, and Drosophila. These changes in GCL activity were detected using saturating levels of substrates, suggesting that V max was dramatically affected, whereas K m values showed no differences. In vitro activation of GCL, presumably due to dephosphorylation, was blocked by inhibitors of protein phosphatases, suggesting that GCL exists in vivo as a mixture of phosphorylated and dephosphorylated forms. The reversibility of the dephosphorylation-dependent activation was indicated by the time-dependent inactivation of the in vitro activated Drosophila GCL, by preincubation with MgATP. NADPH increased maximal GCL activity by up to 93%, whereas several other nucleotide analogues did not, thereby demonstrating specificity. Kinetic analysis using Hanes-Woolf replots of initial velocity data suggested that the NADPH-dependent stimulation of GCL activity is brought about by a change in the maximal activity, V max , rather than changes in substrate affinity. Results of this study suggest that mechanisms of modulation of eukaryotic GCL enzymes may include specific binding of ligands such as pyridine dinucleotide phosphates and reversible protein phosphorylation.
Frontiers in Microbiology
YabT is a serine/threonine kinase of the Hanks family from Bacillus subtilis, which lacks the canonical extracellular signal receptor domain but is anchored to the membrane through a C-terminal transmembrane helix. A previous study demonstrated that a basic juxtamembrane region corresponds to a DNA-binding motif essential for the activation of YabT trans-autophosphorylation. YabT is expressed during spore development and localizes to the asymmetric septum where it specifically phosphorylates essential proteins involved in genome maintenance, such as RecA, SsbA, and YabA. YabT has also been shown to phosphorylate proteins involved in protein synthesis, such as AbrB and Ef-Tu, suggesting a possible regulatory role in the progressive metabolic quiescence of the forespore. Finally, cross phosphorylations with other protein kinases implicate YabT in the regulation of numerous other cellular processes. Using an artificial protein scaffold as crystallization helper, we determined the first crystal structure of this DNA-dependent bacterial protein kinase. This allowed us to trap the active conformation of the kinase domain of YabT. Using NMR, we showed that the basic juxtamembrane region of YabT is disordered in the absence of DNA in solution, just like it is in the crystal, and that it is stabilized upon DNA binding. In comparison with its closest structural homolog, the mycobacterial kinase PknB allowed us to discuss the dimerization mode of YabT. Together with phosphorylation assays and DNA-binding experiments, this structural analysis helped us to gain new insights into the regulatory activation mechanism of YabT.
FEBS Letters, 2004
The ybdL gene of Escherichia coli codes for a protein of unknown function. Sequence analysis showed moderate homology to several vitamin B 6 dependent enzymes, suggesting that it may bind pyridoxal-5 0 -phosphate. The structure analysis of YbdL to 2.35 A resolution by protein crystallography verifies that it is a PLP dependent enzyme of fold type I, the typical aspartate aminotransferase fold. The active site contains a bound pyridoxal-5 0 -phosphate, covalently attached to the conserved active site lysine residue Lys236. The pattern of conserved amino acids in the putative substrate binding pocket of the enzyme reveals that it is most closely related to a hyperthermophilic aromatic residue aminotransferase from the archeon Pyrococcus horikoshii. Activity tests with 10 amino acids as amino-donors reveal, however, a preference for Met, followed by His and Phe, results which can be rationalized by modelization studies.
bioRxiv (Cold Spring Harbor Laboratory), 2023
YisK is an uncharacterized protein in Bacillus subtilis previously shown to interact genetically with the elongasome protein Mbl. YisK overexpression leads to cell widening and lysis, phenotypes that are dependent on mbl and suppressed by mbl mutations. In the present work we characterize YisK's localization, structure, and enzymatic activity. We show that YisK localizes in a punctate and/or punctate-helical pattern that depends on Mbl, and that YisK interacts directly with another elongasome protein, FtsE. YisK belongs to the fumarylacetoacetate hydrolase (FAH) superfamily and crystal structures revealed close structural similarity to two oxaloacetate (OAA) decarboxylases: human mitochondrial FAHD1 and Corynebacterium glutamicum Cg1458. We demonstrate that YisK can also catalyze the decarboxylation of OAA (Km = 134 µM, Kcat = 31 min-1). A catalytic dead variant (YisK E148A, E150A) retains wild-type localization and still widens cells following overexpression, indicating these activities are not dependent on YisK catalysis. Conversely, a non-localizing variant (YisK E30A) retains wild-type enzymatic activity in vitro, but no longer widens cells following overexpression. Together these results suggest YisK may be subject to spatial regulation that depends on the cell envelope synthesis machinery. .
Post-translational regulation and evolution of plant gamma-glutamate cysteine ligase
2007
Glutamate cysteine ligase (GCL) is catalyzing the rate-limiting step in glutathione (GSH) synthesis. A complex regulation of this enzyme is required to integrate various signals as GSH is fulfilling a plethora of functions in housekeeping metabolism, stress defence, and in the regulation of development. In this thesis the post-translational redox regulation of plant GCL and closely related proteobacterial enzymes was studied. The crystal structure of Brassica juncea GCL (BjGCL) revealed the presence of two intramolecular disulfide bridges. Biochemical analyses of the wild-type enzyme and of mutants lacking cysteines required for the formation of either disulfide bridge showed that both bridges are involved in the in vitro redox regulation of BjGCL. One disulfide bridge (CC1) is apparently controlling access to the active site and knock-out results in a slower overall catalysis rate without changes in Km-values. The second disulfide bridge (CC2) controls the formation of a GCL homo-d...
YBX1 (Y box binding protein 1)
Atlas of Genetics and Cytogenetics in Oncology and Haematology, 2012
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence.
YnDL in Complex with 5 amino acid (PGA) complex
2018
The identification of new strategies to fight bacterial infections in view of the spread of multiple resistance to antibiotics has become mandatory. It has been demonstrated that several bacteria develop poly-c-glutamic acid (c-PGA) capsules as a protection from external insults and/or host defence systems. Among the pathogens that shield themselves in these capsules are Bacillus anthracis, Francisella tularensis and several Staphylococcus strains. These are important pathogens with a profound influence on human health. The recently characterised c-PGA hydrolases, which can dismantle the c-PGA-capsules, are an attractive new direction that can offer real hope for the development of alternatives to antibiotics, particularly in cases of multidrug resistant bacteria. We have characterised in detail the cleaving mechanism and stereospecificity of the enzyme PghL (previously named YndL) from Bacillus subtilis encoded by a gene of phagic origin and dramatically efficient in degrading the long polymeric chains of c-PGA. We used X-ray crystallography to solve the three-dimensional structures of the enzyme in its zinc-free, zinc-bound and complexed forms. The protein crystallised with a c-PGA hexapeptide substrate and thus reveals details of the interaction which could explain the stereospecificity observed and give hints on the catalytic mechanism of this class of hydrolytic enzymes.