Mechanistic insights on anserine hydrolyzing activities of human carnosinases.

Anserine and carnosine represent histidine-containing dipeptides that exert a pluripotent protective effect on human physiology. Anserine is known to protect against oxidative stress in diabetes and cardiovascular diseases. Human carnosinases (CN1 and CN2) are dipeptidases involved in the homeostasis of carnosine.

Development of multiple reaction monitoring assay for quantification of carnosine in human plasma.

Carnosine, a histidine containing dipeptide, exerts beneficial effects by scavenging reactive carbonyl compounds (RCCs) that are implicated in pathogenesis of diabetes. However, the reduced carnosine levels may aggravate the severity of diabetes. The precise quantification of carnosine levels may serve as an indicator of pathophysiological state of diabetes.

Two Distinct Assembly States of the Cysteine Regulatory Complex of Salmonella typhimurium Are Regulated by Enzyme-Substrate Cognate Pairs.

Serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS), which catalyze the last two steps of cysteine biosynthesis, interact and form the cysteine regulatory complex (CRC). The current model of Salmonella typhimurium predicts that CRC is composed of one [SAT] unit and two molecules of [OASS]. However, it is not clear why [SAT] cannot engage all of its six high-affinity binding sites.

Structure of soybean serine acetyltransferase and formation of the cysteine regulatory complex as a molecular chaperone.

Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase. Formation of the cysteine regulatory complex (CRC) is a critical biochemical control feature in plant sulfur metabolism.

Equilibrium binding and kinetic characterization of putative tetracycline repressor family transcription regulator Fad35R from Mycobacterium tuberculosis.

Fatty acids play critical role in the survival and virulence of Mycobacterium tuberculosis (Mtb). Activation of fatty acids by acyl-CoA synthetases (Fad) into fatty acyl-CoA is the first and one of the crucial steps in fatty acid metabolism. Mtb possesses 36 fatty acyl-CoA synthetases, unlike Escherichia coli, which has single enzyme.

Second monomer binding is the rate-limiting step in the formation of the dimeric PhoP-DNA complex.

PhoP, the response regulator of the PhoP/PhoQ system, regulates Mg(2+) homeostasis in Salmonella typhimurium. Dimerization of PhoP on the DNA is necessary for its regulatory function, and PhoP regulates the expression of genes in a phosphorylation-dependent manner. Higher PhoP concentrations, however, can activate PhoP and substitute for phosphorylation-dependent gene regulation.

Amino-terminal extension present in the methionine aminopeptidase type 1c of Mycobacterium tuberculosis is indispensible for its activity.

Background: Methionine aminopeptidase (MetAP) is a ubiquitous enzyme in both prokaryotes and eukaryotes, which catalyzes co-translational removal of N-terminal methionine from elongating polypeptide chains during protein synthesis. It specifically removes the terminal methionine in all organisms, if the penultimate residue is non-bulky and uncharged. The MetAP action for exclusion of N-terminal methionine is mandatory in 50-70% of nascent proteins.

Comparative thermodynamic studies on substrate and product binding of O-acetylserine sulfhydrylase reveals two different ligand recognition modes.

Background: The importance of understanding the detailed mechanism of cysteine biosynthesis in bacteria is underscored by the fact that cysteine is the only sulfur donor for all cellular components containing reduced sulfur. O-acetylserine sulfhydrylase (OASS) catalyzes this crucial last step in the cysteine biosynthesis and has been recognized as an important gene for the survival and virulence of pathogenic bacteria. Structural and kinetic studies have contributed to the understanding of mechanistic aspects of OASS, but details of ligand recognition features of OASS are not available.

Cys-Gly specific dipeptidase Dug1p from S. cerevisiae binds promiscuously to di-, tri-, and tetra-peptides: Peptide-protein interaction, homology modeling, and activity studies reveal a latent promiscuity in substrate recognition.

Dug1p is a recently identified novel dipeptidase and plays an important role in glutathione (GSH) degradation. To understand the mechanism of its substrate recognition and specificity towards Cys-Gly dipeptides, we characterized the solution properties of Dug1p and studied the thermodynamics of Dug1p-peptide interactions. In addition, we used homology modeling and ligand docking approaches to get structural insights into Dug1p-peptide interaction.

Assembly of the cysteine synthase complex and the regulatory role of protein-protein interactions.

Macromolecular assemblies play critical roles in regulating cellular functions. The cysteine synthase complex (CSC), which is formed by association of serine O-acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS), acts as a sensor and modulator of thiol metabolism by responding to changes in nutrient conditions. Here we examine the oligomerization and energetics of formation of the soybean CSC.

Contributions of conserved serine and tyrosine residues to catalysis, ligand binding, and cofactor processing in the active site of tyrosine ammonia lyase.

Tyrosine ammonia lyase (TAL) catalyzes the conversion of L-tyrosine to p-coumaric acid using a 3,5-dihydro-5-methylidene-4H-imidazole-4-one (MIO) prosthetic group. In bacteria, TAL is used for production of the photoactive yellow protein chromophore and for caffeic acid biosynthesis in certain actinomycetes. Here we biochemically examine wild-type and mutant forms of TAL from Rhodobacter sphaeroides (RsTAL).

Reaction mechanism of glutathione synthetase from Arabidopsis thaliana: site-directed mutagenesis of active site residues.

Glutathione is essential for maintaining the intracellular redox environment and is synthesized from gamma-glutamylcysteine, glycine, and ATP by glutathione synthetase (GS). To examine the reaction mechanism of a eukaryotic GS, 24 Arabidopsis thaliana GS (AtGS) mutants were kinetically characterized. Within the gamma-glutamylcysteine/glutathione-binding site, the S153A and S155A mutants displayed less than 4-fold changes in kinetic parameters with mutations of Glu-220 (E220A/E220Q), Gln-226 (Q226A/Q226N), and Arg-274 (R274A/R274K) at the distal end of the binding site resulting in 24-180-fold increases in the K(m) values for gamma-glutamylcysteine.

Thermodynamics of the interaction between O-acetylserine sulfhydrylase and the C-terminus of serine acetyltransferase.

Cysteine biosynthesis in plants is partly regulated by the physical association of O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT). Interaction of OASS and SAT requires only the 10 C-terminal residues of SAT. Here we analyze the thermodynamics of formation of a complex of Arabidopsis thaliana OASS (AtOASS) and the C-terminal ligand of AtSAT (C10 peptide) as a function of temperature and salt concentration using fluorescence spectroscopy and isothermal titration calorimetry (ITC).

Structural basis for interaction of O-acetylserine sulfhydrylase and serine acetyltransferase in the Arabidopsis cysteine synthase complex.

In plants, association of O-acetylserine sulfhydrylase (OASS) and Ser acetyltransferase (SAT) into the Cys synthase complex plays a regulatory role in sulfur assimilation and Cys biosynthesis. We determined the crystal structure of Arabidopsis thaliana OASS (At-OASS) bound with a peptide corresponding to the C-terminal 10 residues of Arabidopsis SAT (C10 peptide) at 2.9-A resolution.

Saccharomyces cerevisiae replication protein A binds to single-stranded DNA in multiple salt-dependent modes.

We have examined the single-stranded DNA (ssDNA) binding properties of the Saccharomyces cerevisiae replication protein A (scRPA) using fluorescence titrations, isothermal titration calorimetry, and sedimentation equilibrium to determine whether scRPA can bind to ssDNA in multiple binding modes. We measured the occluded site size for scRPA binding poly(dT), as well as the stoichiometry, equilibrium binding constants, and binding enthalpy of scRPA-(dT)L complexes as a function of the oligodeoxynucleotide length, L. Sedimentation equilibrium studies show that scRPA is a stable heterotrimer over the range of [NaCl] examined (0.

SH2 domains: role, structure and implications for molecular medicine.

Src homology 2 (SH2) domains are protein modules (of approximately 100 amino acids) found in many proteins involved in tyrosine kinase signalling cascades. Their function is to bind tyrosine-phosphorylated sequences in specific protein targets. Binding of an SH2 domain to its cognate tyrosine-phosphorylated target links receptor activation to downstream signalling, both to the nucleus to regulate gene expression and throughout the cytoplasm of the cell.

The tandem Src homology 2 domain of the Syk kinase: a molecular device that adapts to interphosphotyrosine distances.

Conformational flexibility is important for protein function. However, information on the range of conformations accessible to macromolecules in the unbound state is often difficult to obtain. By using the model system of the tandem Src homology 2 domain (i.