Allosteric regulation of substrate channeling: tryptophan synthase.

The regulation of the synthesis of L-tryptophan (L-Trp) in enteric bacteria begins at the level of gene expression where the cellular concentration of L-Trp tightly controls expression of the five enzymes of the Trp operon responsible for the synthesis of L-Trp. Two of these enzymes, trpA and trpB, form an αββα bienzyme complex, designated as tryptophan synthase (TS). TS carries out the last two enzymatic processes comprising the synthesis of L-Trp.

Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate.

NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site.

Discovery of antimicrobial agent targeting tryptophan synthase.

Antibiotic resistance is a continually growing challenge in the treatment of various bacterial infections worldwide. New drugs and new drug targets are necessary to curb the threat of infectious diseases caused by multidrug-resistant pathogens. The tryptophan biosynthesis pathway is essential for bacterial growth but is absent in higher animals and humans.

Mutation of βGln114 to Ala Alters the Stabilities of Allosteric States in Tryptophan Synthase Catalysis.

The tryptophan synthase (TS) bienzyme complexes found in bacteria, yeasts, and molds are pyridoxal 5'-phosphate (PLP)-requiring enzymes that synthesize l-Trp. In the TS catalytic cycle, switching between the open and closed states of the α- and β-subunits via allosteric interactions is key to the efficient conversion of 3-indole-d-glycerol-3'-phosphate and l-Ser to l-Trp. In this process, the roles played by β-site residues proximal to the PLP cofactor have not yet been fully established.

Toho-1 β-lactamase: backbone chemical shift assignments and changes in dynamics upon binding with avibactam.

Backbone chemical shift assignments for the Toho-1 β-lactamase (263 amino acids, 28.9 kDa) are reported based on triple resonance solution-state NMR experiments performed on a uniformly H,C,N-labeled sample. These assignments allow for subsequent site-specific characterization at the chemical, structural, and dynamical levels.

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase.

Structural studies with tryptophan synthase (TS) bienzyme complex (α2β2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated α- and isolated β-subunit allowed the purification of high amounts of pure subunits and α2β2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC).

Structural basis of the XPB helicase-Bax1 nuclease complex interacting with the repair bubble DNA.

Nucleotide excision repair (NER) removes various DNA lesions caused by UV light and chemical carcinogens. The DNA helicase XPB plays a key role in DNA opening and coordinating damage incision by nucleases during NER, but the underlying mechanisms remain unclear. Here, we report crystal structures of XPB from Sulfurisphaera tokodaii (St) bound to the nuclease Bax1 and their complex with a bubble DNA having one arm unwound in the crystal.

Structural and biochemical characterization of a glutathione transferase from the citrus canker pathogen Xanthomonas.

The genus Xanthomonas comprises several cosmopolitan plant-pathogenic bacteria that affect more than 400 plant species, most of which are of economic interest. Citrus canker is a bacterial disease that affects citrus species, reducing fruit yield and quality, and is caused by the bacterium Xanthomonas citri subsp. citri (Xac).

Backbone assignments and conformational dynamics in the S. typhimurium tryptophan synthase α-subunit from solution-state NMR.

Backbone assignments for the isolated α-subunit of Salmonella typhimurium tryptophan synthase (TS) are reported based on triple resonance solution-state NMR experiments on a uniformly H,C,N-labeled sample. From the backbone chemical shifts, secondary structure and random coil index order parameters (RCI-S) are predicted. Titration with the 3-indole-D-glycerol 3'-phosphate analog, N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), leads to chemical shift perturbations indicative of conformational changes from which an estimate of the dissociation constant is obtained.

Structural basis of the XPB-Bax1 complex as a dynamic helicase-nuclease machinery for DNA repair.

Nucleotide excision repair (NER) is a major DNA repair pathway for a variety of DNA lesions. XPB plays a key role in DNA opening at damage sites and coordinating damage incision by nucleases. XPB is conserved from archaea to human.

The copper-inducible copAB operon in Xanthomonas citri subsp. citri is regulated at transcriptional and translational levels.

Upstream open reading frames (ORFs) are frequently found in the 5'-flanking regions of genes and may have a regulatory role in gene expression. A small ORF (named cohL here) was identified upstream from the copAB copper operon in Xanthomonascitri subsp. citri (Xac).

Application of Electrochemical Devices to Characterize the Dynamic Actions of Helicases on DNA.

Much remains to be understood about the kinetics and thermodynamics of DNA helicase binding and activity. Here, we utilize probe-modified DNA monolayers on multiplexed gold electrodes as a sensitive recognition element and morphologically responsive transducer of helicase-DNA interactions. The electrochemical signals from these devices are highly sensitive to structural distortion of the DNA produced by the helicases.

NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity.

Carbanionic intermediates play a central role in the catalytic transformations of amino acids performed by pyridoxal-5'-phosphate (PLP)-dependent enzymes. Here, we make use of NMR crystallography-the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry-to interrogate a carbanionic/quinonoid intermediate analogue in the β-subunit active site of the PLP-requiring enzyme tryptophan synthase. The solid-state NMR chemical shifts of the PLP pyridine ring nitrogen and additional sites, coupled with first-principles computational models, allow a detailed model of protonation states for ionizable groups on the cofactor, substrates, and nearby catalytic residues to be established.

Visualizing the tunnel in tryptophan synthase with crystallography: Insights into a selective filter for accommodating indole and rejecting water.

Four new X-ray structures of tryptophan synthase (TS) crystallized with varying numbers of the amphipathic N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) molecule are presented. These structures show one of the F6 ligands threaded into the tunnel from the β-site and reveal a distinct hydrophobic region. Over this expanse, the interactions between F6 and the tunnel are primarily nonpolar, while the F6 phosphoryl group fits into a polar pocket of the β-subunit active site.

Catalytic roles of βLys87 in tryptophan synthase: (15)N solid state NMR studies.

The proposed mechanism for tryptophan synthase shows βLys87 playing multiple catalytic roles: it bonds to the PLP cofactor, activates C4' for nucleophilic attack via a protonated Schiff base nitrogen, and abstracts and returns protons to PLP-bound substrates (i.e. acid-base catalysis).

Protonation states of the tryptophan synthase internal aldimine active site from solid-state NMR spectroscopy: direct observation of the protonated Schiff base linkage to pyridoxal-5'-phosphate.

The acid-base chemistry that drives catalysis in pyridoxal-5'-phosphate (PLP)-dependent enzymes has been the subject of intense interest and investigation since the initial identification of PLP's role as a coenzyme in this extensive class of enzymes. It was first proposed over 50 years ago that the initial step in the catalytic cycle is facilitated by a protonated Schiff base form of the holoenzyme in which the linking lysine ε-imine nitrogen, which covalently binds the coenzyme, is protonated. Here we provide the first (15)N NMR chemical shift measurements of such a Schiff base linkage in the resting holoenzyme form, the internal aldimine state of tryptophan synthase.

Allostery and substrate channeling in the tryptophan synthase bienzyme complex: evidence for two subunit conformations and four quaternary states.

The allosteric regulation of substrate channeling in tryptophan synthase involves ligand-mediated allosteric signaling that switches the α- and β-subunits between open (low activity) and closed (high activity) conformations. This switching prevents the escape of the common intermediate, indole, and synchronizes the α- and β-catalytic cycles. (19)F NMR studies of bound α-site substrate analogues, N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), were found to be sensitive NMR probes of β-subunit conformation.

Structure of the C-terminal half of human XPB helicase and the impact of the disease-causing mutation XP11BE.

XPB is a DNA-dependent helicase and a subunit of the TFIIH complex required for both transcription and DNA repair. XPB contains four domains: an N-terminal domain, two conserved helicase domains (HD1 and HD2) and a C-terminal extension. The C-terminal extension is important for DNA repair since the phosphorylation of Ser751 inhibits 5'-incision by ERCC1-XPF endonuclease.

The structure of a Xanthomonas general stress protein involved in citrus canker reveals its flavin-binding property.

Xanthomonas citri pv. citri (Xac) causes citrus canker and affects citrus agriculture worldwide. Functional genetic analysis has indicated that a putative general stress protein (XacGSP) encoded by the Xac2369 gene is involved in the bacterial infection.

Production of recombinant EMA-1 protein and its application for the diagnosis of Theileria equi using an enzyme immunoassay in horses from São Paulo State, Brazil.

The erythrocytic-stage surface protein, Equi Merozoite Antigen 1 (EMA-1), is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. In order to establish an effective diagnostic method for practical use, the gene encoding the entire EMA-1 of Theileria equi Jaboticabal strain was cloned and expressed in Escherichia coli as a histidine-tagged protein (His6-EMA1). The expressed EMA-1 reacted with specific antibodies in Western blot and had an apparent molecular mass of 34 kDa which was largely consistent with its theoretical value.

Crystal structures of Xanthomonas small heat shock protein provide a structural basis for an active molecular chaperone oligomer.

Small heat shock proteins (sHsps) are ubiquitous low-molecular-weight chaperones that prevent protein aggregation under cellular stresses. sHsps contain a structurally conserved α-crystallin domain (ACD) of about 100 amino acid residues flanked by varied N- and C-terminal extensions and usually exist as oligomers. Oligomerization is important for the biological functions of most sHsps.

Crystallization and preliminary X-ray diffraction analysis of XAC1151, a small heat-shock protein from Xanthomonas axonopodis pv. citri belonging to the alpha-crystallin family.

The hspA gene (XAC1151) from Xanthomonas axonopodis pv. citri encodes a protein of 158 amino acids that belongs to the small heat-shock protein (sHSP) family of proteins. These proteins function as molecular chaperones by preventing protein aggregation.

Effects of cardiomyopathic mutations on the biochemical and biophysical properties of the human alpha-tropomyosin.

Mutations in the protein alpha-tropomyosin (Tm) can cause a disease known as familial hypertrophic cardiomyopathy. In order to understand how such mutations lead to protein dysfunction, three point mutations were introduced into cDNA encoding the human skeletal tropomyosin, and the recombinant Tms were produced at high levels in the yeast Pichia pastoris. Two mutations (A63V and K70T) were located in the N-terminal region of Tm and one (E180G) was located close to the calcium-dependent troponin T binding domain.

Conditions affecting production of functional muscle recombinant alpha-tropomyosin in Saccharomyces cerevisiae.

Yeasts are attractive hosts for heterologous protein production as they follow the general eukaryotic post-translational modification pattern. The well-known Saccharomyces cerevisiae has been used to produce a large variety of foreign proteins. The proper function of muscle tropomyosin depends on a specific modification at its N-terminus.