Expression, purification and characterization of CTP synthase PyrG in Staphylococcusaureus.

Staphylococcus aureus (S. aureus) presents a significant challenge in both nosocomial and community settings due to its pathogenicity. The emergence of drug-resistant strains exacerbates S. aureus infections, leading to increased mortality rates. PyrG, a member of the cytidine triphosphate (CTP) synthase family, serves as a crucial therapeutic target against S. aureus due to the pivotal role of CTP in cellular metabolism. However, the structural and mechanistic details of S. aureus PyrG remains unknown. Here, we successfully expressed and purified monomeric PyrG. Mutational experiments were conducted based on the results of molecular docking. Based on the results of the molecular docking, we carried out mutation experiments and found that Q386A dramatically decreased the CTP synthase activity compared to the wild-type protein, while Y54A almost completely abolished the activity. Exposure of S. aureus to the kinase inhibitor crizotinib increased expression of gene pyrG. Our results identify the two key sites on PyrG for the CTP synthase activity, and present PyrG gene expression increased during the treatment of crizotinib, which may eventually provide valuable guidance for the development of new drugs against S. aureus infections.

Identification of Interactions in the NMD Complex Using Proximity-Dependent Biotinylation (BioID).

Proximity-dependent trans-biotinylation by the Escherichia coli biotin ligase BirA mutant R118G (BirA*) allows stringent streptavidin affinity purification of proximal proteins. This so-called BioID method provides an alternative to the widely used co-immunoprecipitation (co-IP) to identify protein-protein interactions. Here, we used BioID, on its own and combined with co-IP, to identify proteins involved in nonsense-mediated mRNA decay (NMD), a post-transcriptional mRNA turnover pathway that targets mRNAs that fail to terminate translation properly. In particular, we expressed BirA* fused to the well characterized NMD factors UPF1, UPF2 and SMG5 and detected by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) the streptavidin-purified biotinylated proteins. While the identified already known interactors confirmed the usefulness of BioID, we also found new potentially important interactors that have escaped previous detection by co-IP, presumably because they associate only weakly and/or very transiently with the NMD machinery. Our results suggest that SMG5 only transiently contacts the UPF1-UPF2-UPF3 complex and that it provides a physical link to the decapping complex. In addition, BioID revealed among others CRKL and EIF4A2 as putative novel transient interactors with NMD factors, but whether or not they have a function in NMD remains to be elucidated.

Visualizing Cytoophidia Expression in Drosophila Follicle Cells via Immunohistochemistry.

We describe a user-friendly immunohistochemical approach for the detection of protein localization in Drosophila ovaries, here focusing on CTP synthase. This approach mainly uses fluorescently labeled antibodies to detect single, double, or multiple antigens. We provide a step-by-step protocol with detailed notes and tips, a simplified method that can also be adapted to detect protein localization beyond Drosophila ovaries.

Site-specific biotinylation of purified proteins using BirA.

The binding between biotin and streptavidin or avidin is one of the strongest known non-covalent biological interactions. The (strept)avidin-biotin interaction has been widely used for decades in biological research and biotechnology. Therefore labeling of purified proteins by biotin is a powerful way to achieve protein capture, immobilization, and functionalization, as well as multimerizing or bridging molecules. Chemical biotinylation often generates heterogeneous products, which may have impaired function. Enzymatic biotinylation with E. coli biotin ligase (BirA) is highly specific in covalently attaching biotin to the 15 amino acid AviTag peptide, giving a homogeneous product with high yield. AviTag can conveniently be added genetically at the N-terminus, C-terminus, or in exposed loops of a target protein. We describe here procedures for AviTag insertion by inverse PCR, purification of BirA fused to glutathione-S-transferase (GST-BirA) from E. coli, BirA biotinylation of purified protein, and gel-shift analysis by SDS-PAGE to quantify the extent of biotinylation.

Expression and purification of E. coli BirA biotin ligase for in vitro biotinylation.

The extremely tight binding between biotin and avidin or streptavidin makes labeling proteins with biotin a useful tool for many applications. BirA is the Escherichia coli biotin ligase that site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (also known as Avi-tag). As a complementary approach to in vivo biotinylation of Avi-tag-bearing proteins, we developed a protocol for producing recombinant BirA ligase for in vitro biotinylation. The target protein was expressed as both thioredoxin and MBP fusions, and was released from the corresponding fusion by TEV protease. The liberated ligase was separated from its carrier using HisTrap HP column. We obtained 24.7 and 27.6 mg BirA ligase per liter of culture from thioredoxin and MBP fusion constructs, respectively. The recombinant enzyme was shown to be highly active in catalyzing in vitro biotinylation. The described protocol provides an effective means for making BirA ligase that can be used for biotinylation of different Avi-tag-bearing substrates.

A novel molecular mechanism to explain biotin-unresponsive holocarboxylase synthetase deficiency.

Biotin (vitamins H and B7) is an important micronutrient as defects in its availability, metabolism or adsorption can cause serious illnesses, especially in the young. A key molecule in the biotin cycle is holocarboxylase synthetase (HLCS), which attaches biotin onto the biotin-dependent enzymes. Patients with congenital HLCS deficiency are prescribed oral biotin supplements that, in most cases, reverse the clinical symptoms. However, some patients respond poorly to biotin therapy and have an extremely poor long-term prognosis. Whilst a small number of mutations in the HLCS gene have been implicated, the molecular mechanisms that lead to the biotin-unresponsive phenotype are not understood. To improve our understanding of HLCS, limited proteolysis was performed together with yeast two-hybrid analysis. A structured domain within the N-terminal region that contained two missense mutations was identified in patients who were refractory to biotin therapy, namely p.L216R and p.L237P. Genetic studies demonstrated that the interaction between the enzyme and the protein substrate was disrupted by mutation. Further dissection of the binding mechanism using surface plasmon resonance demonstrated that the mutations reduced affinity for the substrate through a >15-fold increase in dissociation rate. Together, these data provide the first molecular explanation for HLCS-deficient patients that do not respond to biotin therapy.

Biochemical characterization of recombinant guaA-encoded guanosine monophosphate synthetase (EC 6.3.5.2) from Mycobacterium tuberculosis H37Rv strain.

Administration of the current tuberculosis (TB) vaccine to newborns is not a reliable route for preventing TB in adults. The conversion of XMP to GMP is catalyzed by guaA-encoded GMP synthetase (GMPS), and deletions in the Shiguella flexneri guaBA operon led to an attenuated auxotrophic strain. Here we present the cloning, expression, and purification of recombinant guaA-encoded GMPS from Mycobacterium tuberculosis (MtGMPS). Mass spectrometry data, oligomeric state determination, steady-state kinetics, isothermal titration calorimetry (ITC), and multiple sequence alignment are also presented. The homodimeric MtGMPS catalyzes the conversion of XMP, MgATP, and glutamine into GMP, ADP, PP(i), and glutamate. XMP, NH(4)(+), and Mg(2+) displayed positive homotropic cooperativity, whereas ATP and glutamine displayed hyperbolic saturation curves. The activity of ATP pyrophosphatase domain is independent of glutamine amidotransferase domain, whereas the latter cannot catalyze hydrolysis of glutamine to NH(3) and glutamate in the absence of substrates. ITC data suggest random order of binding of substrates, and PP(i) is the last product released. Sequence comparison analysis showed conservation of both Cys-His-Glu catalytic triad of N-terminal Class I amidotransferase and of amino acid residues of the P-loop of the N-type ATP pyrophosphatase family.

A 96-well plate assay for high-throughput analysis of holocarboxylase synthetase activity.

BACKGROUND: Holocarboxylase synthetase (HCS) catalyzes the covalent binding of biotin to both carboxylases and histones. Biotinylated carboxylases and biotinylated histones play crucial roles in the metabolism of fatty acids, amino acids, and glucose, and in gene regulation and genome stability, respectively. HCS null mammals are not viable whereas HCS deficiency is linked to developmental delays in humans and phenotypes such as short life span and low stress resistance in Drosophila. METHODS: HCS-dependent biotinylation of the polypeptide p67 was detected and quantified in a 96-well plate format using IRDye-streptavidin and infrared spectroscopy. RESULTS: Biotinylation of p67 by recombinant HCS (rHCS) and HCS from human cell extracts depended on time, temperature, and substrate concentration, all consistent with enzyme catalysis rather than non-enzymatic biotinylation. The Michaelis-Menten constant of rHCS for p67 was 4.1±1.5 µmol/l. The minimal concentration of rHCS that can be detected by this assay is less than 1.08 nmol/l. Jurkat cells contained 0.14±0.02 U of HCS activity [µmol of biotinylated p67 formed/(nmol/l HCSh)] in 400 µg of total protein. CONCLUSIONS: We developed a 96-well plate assay for high-throughput analysis of HCS activity in biological samples and studies of synthetic and naturally occurring HCS inhibitors.

Expression, purification and analysis of the activity of enzymes from the pentose phosphate pathway.

RNAs, more than ever before, are increasingly viewed as biomolecules of the future, in the versatility of their functions and intricate three-dimensional folding. To effectively study them by nuclear magnetic resonance (NMR) spectroscopy, structural biologists need to tackle two critical challenges of spectral overcrowding and fast signal decay for large RNAs. Stable-isotope nucleotide labeling is one attractive solution to the overlap problem. Hence, developing effective methods for nucleotide labeling is highly desirable. In this work, we have developed a facile and streamlined source of recombinant enzymes from the pentose phosphate pathway for making such labeled nucleotides. The Escherichia coli (E. coli) genes encoding ribokinase (RK), adenine phosphoribosyltransferase (APRT), xanthine/guanine phosphoribosyltransferase (XGPRT), and uracil phosphoribosyltransferase (UPRT) were sub-cloned into pET15b vectors. All four constructs together with cytidine triphosphate synthetase (CTPS) and human phosphoribosyl pyrophosphate synthetase isoform 1 (PRPPS) were transformed into the E. coli BL21(AI) strain for protein over-expression. The enzyme preparations were purified to >90% homogeneity by a one-step Ni-NTA affinity chromatography, without the need of a further size-exclusion chromatography step. We obtained yields of 1530, 22, 482, 3120, 2120 and 2280 units of activity per liter of culture for RK, PRPPS, APRT, XGPRT, UPRT and CTPS, respectively; the specific activities were found to be 70, 22, 21, 128, 144 and 113 U/mg, respectively. These specific activities of these enzyme constructs are comparable to or higher than those previously reported. In addition, both the growth conditions and purification protocols have been streamlined so that all the recombinant proteins can be expressed, purified and characterized in at most 2 days. The availability and reliability of these constructs should make production of fully and site-specific labeled nucleotides for making labeled RNA accessible and straightforward, to facilitate high-resolution NMR spectroscopic and other biophysical studies.

Generation of peptide MHC class I monomers and multimers through ligand exchange.

The recognition of defined antigen-MHC complexes by antigen-specific T cells forms the molecular basis of T cell immunity. It has been shown that fluorescently labeled recombinant MHC tetramers can be utilized to detect antigen-specific T cells by flow cytometry. Since this first description, MHC tetramers and other types of MHC multimers have become a core tool to monitor the development of disease- and therapy-induced antigen-specific T cell responses both in humans and in animal model systems. This unit describes a set of protocols that transform classical MHC multimer technology into a high-throughput platform, allowing one to produce large collections of MHC class I molecules charged with different peptides. This technology is based on the development of conditional MHC ligands that can be triggered to self-destruct while in the MHC-bound state.

Distinct amino termini of two human HCS isoforms influence biotin acceptor substrate recognition.

The human holocarboxylase synthetase (HCS) catalyzes transfer of biotin to biotin-dependent carboxylases, and the enzyme is therefore of fundamental importance for many physiological processes, including fatty acid synthesis, gluconeogenesis, and amino acid catabolism. In addition, the enzyme functions in regulating transcription initiation at several genes that code for proteins involved in biotin metabolism. Two major forms of HCS exist in humans, which differ at the amino terminus by 57 amino acids. In this work, the two proteins were expressed in Escherichia coli, purified, and subjected to biochemical characterization. Equilibrium sedimentation indicates that the two proteins are monomers both in their apo-forms and when bound to the enzymatic intermediate biotinyl 5'-AMP. Steady state kinetic analyses as a function of biotin, ATP, or a minimal biotin-accepting substrate concentration indicate similar behaviors for both isoforms. However, pre-steady state analysis of biotin transfer reveals that the full-length HCS associates with the minimal biotin acceptor substrate with a rate twice as fast as that of the truncated isoform. These results are consistent with a role for the HCS amino terminus in biotin acceptor substrate recognition.

A biotin-based protein tagging system.

Biotin protein ligase (BPL) mediates covalent attachment of biotin to a specific lysine residue of biotin carboxyl carrier protein (BCCP) of biotin-dependent enzymes. We recently found that the biotinylation reaction from thermophilic archaeon Sulfolobus tokodaii has a unique characteristic that the enzyme BPL forms a tight complex with the product, biotinylated BCCP (169 amino acid residues). In the current work, we attempted to apply this characteristic to a novel protein tagging system. Thus, the N terminus of S. tokodaii BCCP was truncated and the interaction of the resulting BCCP, BCCPDelta100 and BCCPDelta17 (with 69 and 152 residues, respectively), with BPL was investigated by surface plasmon resonance (SPR). It was found that the binding of BPL to the biotinylated BCCPDelta100 is extremely tight with a dissociation constant (K(D)) of 1.2 nM, whereas that to the unbiotinylated counterpart was moderate with a K(D) of 3.3 microM. Furthermore, chimeric proteins of glutathione S-transferase (GST) and green fluorescence protein (GFP) with BCCPDelta100 fused to their C terminus were prepared. The resulting fusion proteins were successfully biotinylated and captured on the BPL-modified SPR sensor chip or BPL-modified magnetic beads. The function of GST and GFP was hardly impaired on fusion with BCCPDelta100 and biotinylation of the latter.

Purification, crystallization and preliminary crystallographic analysis of biotin protein ligase from Staphylococcus aureus.

Biotin protein ligase from Staphylococcus aureus catalyses the biotinylation of acetyl-CoA carboxylase and pyruvate carboxylase. Recombinant biotin protein ligase from S. aureus has been cloned, expressed and purified. Crystals were grown using the hanging-drop vapour-diffusion method using PEG 8000 as the precipitant at 295 K. X-ray diffraction data were collected to 2.3 A resolution from crystals using synchrotron X-ray radiation at 100 K. The diffraction was consistent with the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 93.665, c = 131.95.

High-yield expression and purification of isotopically labeled norcoclaurine synthase, a Bet v 1-homologous enzyme, from Thalictrum flavum for NMR studies.

The enzyme norcoclaurine synthase (NCS) found in the common meadow rue, Thalictrum flavum, and other plants shows sequence homology to members of the class 10 of pathogenesis related (PR 10) proteins that contains allergens such as the major birch pollen allergen Bet v 1, the major cherry allergen Pru av 1, and the major apple allergen Mal d 1. The enzyme is involved in the plant's secondary metabolism and is required for the production of bioactive secondary metabolites like morphine. Whereas the physiological function of PR 10 class allergens is still unknown, NCS activity has been studied in detail. Investigation of the structural properties of NCS by NMR spectroscopy can thus not only provide new information concerning the reaction mechanism of the enzyme, but is also expected to help clarify the long standing and heavily debated question on the physiological function as well as the reasons for the allergenic potential of members of this protein family. As the first important step towards the three-dimensional solution structure, we optimized expression of recombinant NCS in Escherichia coli and established an efficient purification protocol yielding high amounts of pure isotopically labeled active enzyme. The identity of NCS was confirmed by electrospray ionization mass spectrometry, and activity of the purified enzyme was determined by an assay detecting the radiolabeled reaction product. Spectroscopic analysis by NMR spectroscopy showed that the protein was properly folded with well defined tertiary structure.

Mechanistic studies on norcoclaurine synthase of benzylisoquinoline alkaloid biosynthesis: an enzymatic Pictet-Spengler reaction.

Norcoclaurine synthase catalyzes an asymmetric Pictet-Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde to give (S)-norcoclaurine. This is the first committed step in the biosynthesis of the benzylisoquinoline alkaloids that include morphine and codeine. In this work, the gene encoding for the Thalictrum flavum norcoclaurine synthase is highly overexpressed in Escherichia coli and the resulting His-tagged recombinant enzyme is purified for the first time. A continuous assay based on circular dichroism spectroscopy is developed and used to monitor the kinetics of the enzymatic reaction. Dopamine analogues bearing a methoxy or hydrogen substituent in place of the C-1 phenolic group were readily accepted by the enzyme whereas those bearing the same substituents at C-2 were not. This supports a mechanism involving a two-step cyclization of the putative iminium ion intermediate that does not proceed via a spirocyclic intermediate. The reaction of [3,5,6-2H]dopamine was found to be slowed by a kinetic isotope effect of 1.7 +/- 0.1 on the value of kcat/KM. This is interpreted as showing that the deprotonation step causing rearomatization is partially rate determining in the overall reaction.

Rate-limiting steps and role of active site Lys443 in the mechanism of carbapenam synthetase.

Carbapenam synthetase (hereafter named CPS) catalyzes the formation of the beta-lactam ring in the biosynthetic pathway to (5R)-carbapen-2-em-3-carboxylate, the simplest of the carbapenem antibiotics. Kinetic studies showed remarkable tolerance to substrate stereochemistry in the turnover rate but did not distinguish between chemistry and a nonchemical step such as product release or conformational change as being rate-determining. Also, X-ray structural studies and modest sequence homology to beta-lactam synthetase, an enzyme that catalyzes the formation of a monocyclic beta-lactam ring in a similar ATP/Mg2+-dependent reaction, implicate K443 as an essential residue for substrate binding and intermediate stabilization. In these experiments, we use pH-rate profiles, deuterium solvent isotope effects, and solvent viscosity measurements to examine the rate-limiting step in this complex overall process of substrate adenylation and intramolecular ring formation. Mutagenesis and chemical rescue demonstrate that K443 is the general acid visible in the pH-rate profile of the wild-type CPS-catalyzed reaction. On the basis of these results, we propose a mechanism in which the rate-limiting step is beta-lactam ring formation coupled to a protein conformational change and underscore the role of K443 throughout the reaction.

Structure of 5-formyltetrahydrofolate cyclo-ligase from Bacillus anthracis (BA4489).

Bacillus anthracis is a spore-forming bacterium and the causative agent of the disease anthrax. The Oxford Protein Production Facility has been targeting proteins from B. anthracis in order to develop high-throughput technologies within the Structural Proteomics in Europe project. As part of this work, the structure of 5-formyltetrahydrofolate cyclo-ligase (BA4489) has been determined by X-ray crystallography to 1.6 A resolution. The structure, solved in complex with magnesium-ion-bound ADP and phosphate, gives a detailed picture of the proposed catalytic mechanism of the enzyme. Chemical differences from other cyclo-ligase structures close to the active site that could be exploited to design specific inhibitors are also highlighted.

Functional analysis of norcoclaurine synthase in Coptis japonica.

(S)-Norcoclaurine is the entry compound in benzylisoquinoline alkaloid biosynthesis and is produced by the condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) by norcoclaurine synthase (NCS) (EC 4.2.1.78). Although cDNA of the pathogenesis-related (PR) 10 family, the translation product of which catalyzes NCS reaction, has been isolated from Thalictrum flavum, its detailed enzymological properties have not yet been characterized. We report here that a distinct cDNA isolated from Coptis japonica (CjNCS1) also catalyzed NCS reaction as well as a PR10 homologue of C. japonica (CjPR10A). Both recombinant proteins stereo-specifically produced (S)-norcoclaurine by the condensation of dopamine and 4-HPAA. Because a CjNCS1 cDNA that encoded 352 amino acids showed sequence similarity to 2-oxoglutarate-dependent dioxygenases of plant origin, we characterized the properties of the native enzyme. Sequence analysis indicated that CjNCS1 only contained a Fe(2+)-binding site and lacked the 2-oxoglutarate-binding domain. In fact, NCS reaction of native NCS isolated from cultured C. japonica cells did not depend on 2-oxoglutarate or oxygen, but did require ferrous ion. On the other hand, CjPR10A showed no specific motif. The addition of o-phenanthroline inhibited NCS reaction of both native NCS and recombinant CjNCS1, but not that of CjPR10A. In addition, native NCS and recombinant CjNCS1 accepted phenylacetaldehyde and 3,4-dihydroxyphenylacetaldehyde, as well as 4-HPAA, for condensation with dopamine, whereas recombinant CjPR10A could use 4-hydroxyphenylpyruvate and pyruvate in addition to the above aldehydes. These results suggested that CjNCS1 is the major NCS in C. japonica, whereas native NCS extracted from cultured C. japonica cells was more active and formed a larger complex compared with recombinant CjNCS1.

The effects of removing the GAT domain from E. coli GMP synthetase.

E. coli GMP synthetase (GMPS) catalyzes the conversion of XMP to GMP. Ammonia, generated in the amino-terminal glutamine amidotransferase (GAT) domain, is transferred by an unknown mechanism to the ATP-pyrophosphatase (ATPP) domain, where it attacks a highly reactive adenyl-XMP intermediate, leading to GMP formation. To study the structural requirements for the activity of E. coli GMPS, we used PCR to generate a protein expression construct that contains the ATPP domain as well as the predicted dimerization domain (DD). The ATPP/DD protein is active in solution, utilizing NH (4) (+) as an NH(3) donor. Size-exclusion chromatography demonstrates a dimeric mass for the ATPP/ DD protein, providing the first evidence in solution for the structural organization of the intact GMPS. Kinetic characterization of the ATPP/DD domain protein provides evidence that the presence of the GAT domain can regulate the activity of the ATPP domain.

Structure of the synthetase domain of human CTP synthetase, a target for anticancer therapy.

Cytidine triphosphate synthetase (CTPS) is a key enzyme in nucleic acid and phospholipid biosynthesis and its activity is increased in certain human cancers, making it a promising drug target. The crystal structure of the synthetase domain of human CTPS, which represents the first structure of a CTPS from an eukaryote, has been determined. The structure is homotetrameric and each active site is formed by three different subunits. Sulfate ions bound to the active sites indicate the positions of phosphate-binding sites for the substrates ATP and UTP and the feedback inhibitor CTP. Together with earlier structures of bacterial CTPS, the human CTPS structure provides an extended understanding of the structure-function relationship of CTPS-family members. The structure also serves as a basis for structure-based design of anti-proliferative inhibitors.