Role of Pyridoxal 5′-Phosphate in the Structural Stabilization of O-Acetylserine Sulfhydrylase

Self Cite

Section: Effects Of Tryptophan Substitutions On the Secondary Structumentioning

confidence: 74%

“…A low thermodynamic stability was evidenced for holo-OASS (6). Most of the stabilization free energy was demonstrated to derive from the binding of the cofactor to the active site, with a more pronounced effect on the C-terminal domain (7), which appears to be more stable than the N-terminal domain.…”

mentioning

confidence: 98%

Surface-exposed Tryptophan Residues Are Essential for O-Acetylserine Sulfhydrylase Structure, Function, and Stability

Campanini

Raboni

Vaccari

et al. 2003

Self Cite

“…PLP-induced stabilization has been observed previously for many, but not all, PLP-binding enzymes. For example, rat liver mitochondrial aspartate aminotransferase, O-acetylserine sulfhydrylase, glutamate decarboxylase, sheep liver serine hydroxymethyltransferase, and tryptophan synthase were found to be stabilized by PLP (33)(34)(35)(36)(37)(38), but no stabilization has been observed for serine hydroxymethyltransferase (39) or dopa decarboxylase (40).…”

Section: Discussionmentioning

confidence: 99%

In Vivo and in Vitro Examination of Stability of Primary Hyperoxaluria-associated Human Alanine:Glyoxylate Aminotransferase

Hopper

Pittman

Fitzgerald

et al. 2008

Primary hyperoxaluria type I is a severe kidney stone disease caused by mutations in the protein alanine:glyoxylate aminotransferase (AGT). Many patients have mutations in AGT that are not deleterious alone but act synergistically with a common minor allele polymorphic variant to impair protein folding, dimerization, or localization. Although studies suggest that the minor allele variant itself is destabilized, no direct stability studies have been carried out. In this report, we analyze AGT function and stability using three approaches. First, we describe a yeast complementation growth assay for AGT, in which we show that human AGT can substitute for function of yeast Agx1 and that mutations associated with disease in humans show reduced growth in yeast. The reduced growth of minor allele mutants reflects reduced protein levels, indicating that these proteins are less stable than wild-type AGT in yeast. We further examine stability of AGT alleles in vitro using two direct methods, a mass spectrometry-based technique (stability of unpurified proteins from rates of H/D exchange) and differential scanning fluorimetry. We also examine the effect of known ligands pyridoxal 5-phosphate and aminooxyacetic acid on stability. Our work establishes that the minor allele is destabilized and that pyridoxal 5-phosphate and aminooxyacetic acid binding significantly stabilizes both alleles. To our knowledge, this is the first work that directly measures relative stabilities of AGT variants and ligand complexes. Because previous studies suggest that stabilizing compounds (i.e. pharmacological chaperones) may be effective for treatment of primary hyperoxaluria, we propose that the methods described here can be used in high throughput screens for compounds that stabilize AGT mutants.Deficiencies in the enzyme alanine:glyoxylate aminotransferase (AGT) 3 cause primary hyperoxaluria type I (PH1), a severe autosomal recessive kidney stone disease (1, 2). In humans, AGT is responsible for conversion of glyoxylate to glycine in the liver. Without functional AGT, glyoxylate builds up and is converted to calcium oxalate, which is deposited in the kidneys and can lead to kidney stones and renal failure. In many patients, deficiency of AGT results from one or two amino acid changes that decrease the stability of this enzyme. As a result, AGT may be degraded, become improperly localized, or form nonfunctional aggregates (1, 2).Over 50 different mutations of AGT and two polymorphic variants have been identified (1, 3). The two allelic forms consist of a "wild-type" major allele, AGTma, and a minor allele, AGTmi. The minor allele is present in ϳ20% of European and North American populations and contains P11L and I340M substitutions in the amino acid sequence and a 74-bp duplication in intron 1 (4, 5). The P11L and I340M substitutions, particularly P11L, have several biochemical effects, including decreasing catalytic activity and slowing the dimerization rate, but by themselves are not disease-causing (4, 5). The minor allele of AGT is delete...

“…As in the case of the CysE-CysK complex, OAS might induce the dissociation of the CymR-CysK complex either by directly competing with CymR for binding to the active site of CysK or by altering the conformation of CysK indirectly preventing its interaction with CymR. PLP has been reported to stabilize the native fold of most of PLP enzymes (47). In the case of MalY, PLP is also required to maintain it in a conformation capable of interacting with its associated regulator MalT (41,48).…”

Section: Discussionmentioning

confidence: 99%

The CymR Regulator in Complex with the Enzyme CysK Controls Cysteine Metabolism in Bacillus subtilis

Tanous

Soutourina

Raynal

et al. 2008

108

Several enzymes have evolved as sensors in signal transduction pathways to control gene expression, thereby allowing bacteria to adapt efficiently to environmental changes. We recently identified the master regulator of cysteine metabolism in Bacillus subtilis, CymR, which belongs to the poorly characterized Rrf2 family of regulators. We now report that the signal transduction mechanism controlling CymR activity in response to cysteine availability involves the formation of a stable complex with CysK, a key enzyme for cysteine biosynthesis. We carried out a comprehensive quantitative characterization of this regulator-enzyme interaction by surface plasmon resonance and analytical ultracentrifugation. We also showed that O-acetylserine plays a dual role as a substrate of CysK and as an effector modulating the CymR-CysK complex formation. The ability of B. subtilis CysK to bind to CymR appears to be correlated to the loss of its capacity to form a cysteine synthase complex with CysE. We propose an original model, supported by the determination of the intracellular concentrations of the different partners, by which CysK positively regulates CymR in sensing the bacterial cysteine pool.