Hexahistidine Fusion Research Articles

Recombinant Surface Proteomics as a Tool to Analyze Humoral Immune Responses in Bovines Infected by Mycoplasma mycoides Subsp. mycoides Small Colony Type

A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causative agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented. Humoral immune responses in 242 CBPP-affected cattle and controls were monitored against one-third of the surface proteins of M. mycoides SC in a high throughput magnetic bead-based assay. Initially, 64 surface proteins were selected from the genome sequence of M. mycoides SC and expressed as recombinant proteins in Escherichia coli. Binding of antibodies to each individual protein could then be analyzed simultaneously in minute sample volumes with the Luminex suspension array technology. The assay was optimized on Namibian CBPP-positive sera and Swedish negative controls to allow detection and 20-fold mean signal separation between CBPP-positive and -negative sera. Signals were proven to be protein-specific by inhibition experiments, and results agreed with Western blot experiments. The potential of the assay to monitor IgG, IgM, and IgA responses over time was shown in a proof-of-concept study with 116 sera from eight animals in a CBPP vaccine study. In conclusion, a toolbox with recombinant proteins and a flexible suspension array assay that allows multiplex analysis of humoral immune responses to M. mycoides SC has been created.

Heterologous expression and molecular characterization of the NAD(P)H:acceptor oxidoreductase (FerB) of Paracoccus denitrificans

FerB is a flavoenzyme capable of reducing quinones, ferric complexes and chromate. Its expression in Escherichia coli as a hexahistidine fusion resulted in a functional product only when the tag was placed on the C-terminus. The molecular mass values estimated by gel permeation chromatography were compatible with the existence of either dimer or trimer, whereas the light scattering data, together with cross-linking experiments that yielded exclusively monomer and dimer bands on dodecyl sulfate–polyacrylamide gels, strongly supported a dimeric nature of both native and tagged form of FerB. These two proteins also exhibited almost identical secondary structure as judged by Fourier transform infra red spectrometry. The presence of tag, however, shifted the temperature of thermal inactivation as well as the thermal denaturation curve towards lower temperatures. Despite somewhat lower thermal stability, the fusion protein is considered a better candidate for crystallization than the wild-type one due to a more negative value of its second optical virial coefficient.

Separation of hexahistidine fusion proteins with immobilized metal ion affinity chromatographic (IMAC) sorbents derived from MN+‐tacn and its derivatives

The capabilities of a new class of immobilized (im) metal ion chelate complexes (IMCCs), derived from 1,4,7-triazacyclononane (tacn), bis(1,4,7-triazacyclononyl) ethane (dtne) and bis(1,4,7-triazacyclononyl)propane (dtnp) complexed with the borderline metal ions Cu(2+), Ni(2+), Zn(2+), Mn(2+), Co(2+), and Cr(3+), for the purification of proteins have been investigated. In particular, the binding behavior of a model protein, the C-terminal hexahistidine tagged recombinant fusion protein Schistosoma japonicum glutathione S-transferase-Saccharomyces cerevisiae mitochondrial ATP synthase delta-subunit (GST-deltaATPase-His(6)), with these new immobilized metal ion affinity chromatographic (IMAC) sorbents was compared to the properties of a conventional sorbent, derived from immobilized Ni(II)-nitrilotriacetic acid (im-Ni(2+)-NTA). Investigations using the recombinant GST-deltaATPase-His(6) and recombinant S. japonicum glutathione S-transferase (GST) lacking a hexahistidine tag have confirmed that the C-terminal tag hexahistidine residues were required for the binding process to occur with these IMAC systems. The results also confirm that recombinant fusion proteins such as GST-deltaATPase-His(6) can be isolated in high purity with these IMAC systems. Moreover, these new macrocyclic systems manifest different selectivity features as a function of pH or ionic strength when compared to the conventional, unconstrained iminodiacetic acid (IDA) or NTA chelating ligands, complexed with borderline metal ions such as Cu(2+) or Ni(2+), as IMAC systems.

Purification of Caudal-Related Homeodomain Transcription Factor and Its Binding Characterization

Human CDX2 is known as a caudal-related homeodomain transcription factor that is expressed in the intestinal epithelium and is important in differentiation and maintenance of the intestinal epithelial cells. The caudal-related homeobox proteins bind DNA according to a helix-turn-helix structure, thereby increasing the structural stability of DNA. A cancer-tumor suppressor role for Cdx2 has been shown by a decrease in the level of the expression of Cdx2 in colorectal cancer but the mechanism of transcriptional regulation has not been examined at the molecular level. We developed a large-scale system for expression of the recombinant, novel CDX2, in the Escherichia coli. A highly purified and soluble CDX2 protein was obtained in E. coli strain BL21(DE3)RIL and a hexahistidine fusion system using Ni-NTA affinity column, anion exchange, and gel filtration chromatography. The identity and secondary structure of the purified CDX2 protein were confirmed by MALDI-TOF MS, Western blot, and a circular dichroism analyses. In addition, we studied the DNA binding activity of recombinant CDX2 by ELISA experiment and isolated human CDX2 binding proteins derived from rat cells by an immobilized GST-fusion method. Three CDX2-binding proteins were found in the gastric tissue, and those proteins were identified to the homeobox protein Hox-D8, LIM homeobox protein 6, and SMC1L1 protein.

IRBIT, Inositol 1,4,5-Triphosphate (IP3) Receptor-binding Protein Released with IP3, Binds Na+/H+ Exchanger NHE3 and Activates NHE3 Activity in Response to Calcium

Calcium (Ca2+) is a highly versatile second messenger that regulates various cellular processes. Previous studies showed that elevation of intracellular Ca2+ regulates the activity of Na+/H+ exchanger 3 (NHE3). However, the effect of Ca2+-dependent signaling on NHE3 activity varies depending on cell types. In this study, we report the identification of IP3 receptor-binding protein released with IP3 (IRBIT) as a NHE3 interacting protein and its role in regulation of NHE3 activity. IRBIT bound to the carboxyl-terminal domain of NHE3, which is necessary for acute regulation of NHE3. Ectopic expression of IRBIT resulted in Ca2+-dependent activation of NHE3 activity, whereas silencing of endogenous IRBIT resulted in inhibition of NHE3 activity. Ca2+-dependent stimulation of NHE3 activity was dependent on the binding of IRBIT to NHE3. Previously Ca2+-dependent inhibition of NHE3 was demonstrated in the presence of NHERF2. Co-expression of IRBIT was able to reverse the NHERF2-dependent inhibition of NHE3. We also showed that IRBIT-dependent activation of NHE3 involves exocytic trafficking of NHE3 to the plasma membrane and this activation was blocked by inhibition of calmodulin (CaM) or CaM-dependent kinase II. These results suggest that the overall effect of Ca2+ on NHE3 activity is balanced by IRBIT-dependent activation and NHERF2-dependent inhibition.

Cloning, expression, purification and preliminary crystallographic analysis of the RNase HI domain of the Mycobacterium tuberculosis protein Rv2228c as a maltose-binding protein fusion.

The predicted ribonuclease (RNase) HI domain of the open reading frame Rv2228c from Mycobacterium tuberculosis has been cloned as a hexahistidine fusion and a maltose-binding protein (MBP) fusion. Expression was only observed for the MBP-fusion protein, which was purified using amylose affinity chromatography and gel filtration. The RNase HI domain could be cleaved from the MBP-fusion protein by factor Xa digestion, but was very unstable. In contrast, the fusion protein was stable, could be obtained in high yield and gave crystals which diffracted to 2.25 A resolution. The crystals belong to space group P2(1) and have unit-cell parameters a = 73.63, b = 101.38, c = 76.09 A, beta = 109.0 degrees. Two fusion-protein molecules of 57,417 Da were present in each asymmetric unit.

Protein Kinase C Phosphorylation Disrupts Na+/H+ Exchanger Regulatory Factor 1 Autoinhibition and Promotes Cystic Fibrosis Transmembrane Conductance Regulator Macromolecular Assembly

An emerging theme in cell signaling is that membrane-bound channels and receptors are organized into supramolecular signaling complexes for optimum function and cross-talk. In this study, we determined how protein kinase C (PKC) phosphorylation influences the scaffolding protein Na(+)/H(+) exchanger regulatory factor 1 (NHERF) to assemble protein complexes of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel that controls fluid and electrolyte transport across cell membranes. NHERF directs polarized expression of receptors and ion transport proteins in epithelial cells, as well as organizes the homo- and hetero-association of these cell surface proteins. NHERF contains two modular PDZ domains that are modular protein-protein interaction motifs, and a C-terminal domain. Previous studies have shown that NHERF is a phosphoprotein, but how phosphorylation affects NHERF to assemble macromolecular complexes is unknown. We show that PKC phosphorylates two amino acid residues Ser-339 and Ser-340 in the C-terminal domain of NHERF, but a serine 162 of PDZ2 is specifically protected from being phosphorylated by the intact C-terminal domain. PKC phosphorylation-mimicking mutant S339D/S340D of NHERF has increased affinity and stoichiometry when binding to C-CFTR. Moreover, solution small angle x-ray scattering indicates that the PDZ2 and C-terminal domains contact each other in NHERF, but such intramolecular domain-domain interactions are released in the PKC phosphorylation-mimicking mutant indicating that PKC phosphorylation disrupts the autoinhibition interactions in NHERF. The results demonstrate that the C-terminal domain of NHERF functions as an intramolecular switch that regulates the binding capability of PDZ2, and thus controls the stoichiometry of NHERF to assemble protein complexes.

Characterization of hbzE-encoded gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867

Pseudomonas alcaligenes NCIMB 9867 (strain P25X) is known to synthesize two isofunctional gentisate 1,2-dioxygenases (GDO; EC 1.13.11.4) as well as other enzymes involved in the degradation of xylenols and cresols via the gentisate pathway. The hbzE gene encoding what is possibly the strictly inducible gentisate 1,2-dioxygenase II (GDO-II) was cloned, overexpressed and purified as a hexahistidine fusion protein from Escherichia coli. Active recombinant GDO-II had an estimated molecular mass of 150 kDa and is likely a tetrameric protein with a subunit mass of ∼40 kDa, similar to the previously characterized gentisate 1,2-dioxygenase I (GDO-I) encoded by xlnE. However, GDO-II was unable to utilize gentisate that is substituted at the carbon-4 position, unlike GDO-I which had broader substrate specificity. GDO-II also possessed different kinetic characteristics when compared to GDO-I. The hbzE-encoded GDO-II shared higher sequence identities (53%) with GDOs from Ralstonia sp. U2 and Polaromonas naphthalenivorans CJ2, compared with only 35% identity with the xlnE-encoded GDO-I. The hbzE gene was found to be part of a cluster of nine genes including the putative regulatory gene designated hbzR, which encodes an LysR-type regulator and is divergently transcribed from the other genes of the hbzHIJKLFED cluster.

Molecular and Structural Characterization of the PezAT Chromosomal Toxin-Antitoxin System of the Human Pathogen Streptococcus pneumoniae

The chromosomal pezT gene of the Gram-positive pathogen Streptococcus pneumoniae encodes a protein that is homologous to the zeta toxin of the Streptococcus pyogenes plasmid pSM19035-encoded epsilon-zeta toxin-antitoxin system. Overexpression of pezT in Escherichia coli led to severe growth inhibition from which the bacteria recovered approximately 3 h after induction of expression. The toxicity of PezT was counteracted by PezA, which is encoded immediately upstream of pezT and shares weak sequence similarities in the C-terminal region with the epsilon antitoxin. The pezAT genes form a bicistronic operon that is co-transcribed from a sigma(70)-like promoter upstream of pezA and is negatively autoregulated with PezA functioning as a transcriptional repressor and PezT as a co-repressor. Both PezA and the non-toxic PezA(2)PezT(2) protein complex bind to a palindrome sequence that overlaps the promoter. This differs from the epsilon-zeta system in which epsilon functions solely as the antitoxin and transcriptional regulation is carried out by another protein designated omega. Results from site-directed mutagenesis experiments demonstrated that the toxicity of PezT is dependent on a highly conserved phosphoryltransferase active site and an ATP/GTP nucleotide binding site. In the PezA(2)PezT(2) complex, PezA neutralizes the toxicity of PezT by blocking the nucleotide binding site through steric hindrance.

Branched‐Chain Keto Acid Decarboxylase from Lactococcus lactis (KdcA), a Valuable Thiamine Diphosphate‐Dependent Enzyme for Asymmetric CC Bond Formation

AbstractThe thiamine diphosphate‐dependent, branched‐chain 2‐keto acid decarboxylase from Lactococcus lactis sup. cremoris B1157 (KdcA) is a new valuable enzyme for the synthesis of chiral 2‐hydroxy ketones. The gene was cloned and the enzyme was expressed as an N‐terminal hexahistidine fusion protein in Escherichia coli. It has a broad substrate range for the decarboxylation reaction including linear and branched‐chain aliphatic and aromatic keto acids as well as phenyl pyruvate and indole‐3‐pyruvate. The dimeric structure of recombinant KdcA is in contrast to the tetrameric structure of other 2‐keto acid decarboxylases. The enzyme is stable between pH 5 and 7 with a pH optimum of pH 6–7 for the decarboxylation reaction. While KdcA is sufficiently stable up to 40 °C it rapidly looses activity at higher temperatures. In this work the carboligase activity of KdcA is demonstrated for the first time. The enzyme shows an exceptionally broad substrate range and, most strikingly, it catalyzes the carboligation of different aromatic aldehydes as well as CH‐acidic aldehydes such as phenylacetaldehyde and indole‐3‐acetaldehyde with aliphatic aldehydes such as acetaldehyde, propanal, and cyclopropanecarbaldehyde, yielding chiral 2‐hydroxy ketones in high enantiomeric excess. Noteworthy, the donor‐acceptor selectivity is strongly influenced by the nature of the respective substrate combination.

The repairing effect of a recombinant human connective‐tissue growth factor in a burn‐wounded rhesus‐monkey (Macaca mulatta) model

CTGF (connective-tissue growth factor) has been characterized as an extracellular-matrix-associated protein that modulates basic-fibroblast-growth-factor signalling and angiogenesis. In the present paper, the cloning of the ctgf gene from human umbilical-vein endothelial cells and expression of the protein in Escherichia coli as an N-terminal hexahistidine fusion protein is described. Recombinant human CTGF (rhCTGF) was expressed and purified so that we could investigate its effect on the proliferation of human embryo fibroblast KMB-17 and NIH3T3 cells. The results indicated not only that the protein was properly folded, but also that it had the same specific activity and stability as the native protein. Furthermore, we administered this recombinant protein in a non-human primate [rhesus monkey (Macaca mulatta)] burn-wound model and report the clinical findings and structural effects. Epitheliotrophic effects were conspicuous in wounded tissues at 10-100 ng of CTGF/cm(2), suggesting that administered rhCTGF can play a normal physiological role in wound repairing in a non-human primate model.

Biochemical Characterization of a Regulatory Cascade Controlling Transcription of the Pseudomonas aeruginosa Type III Secretion System

Many Gram-negative pathogens utilize type III secretion systems (T3SS) to translocate effector proteins into eukaryotic host cells. Expression of T3SS genes is highly regulated and is often coupled to type III secretory activity. Transcription of the Pseudomonas aeruginosa T3SS genes is coupled to secretion by a cascade of interacting regulatory proteins (ExsA, ExsD, ExsC, and ExsE). ExsA is an activator of type III gene transcription, ExsD binds ExsA to inhibit transcription, ExsC inhibits ExsD activity, and ExsE inhibits ExsC activity. The entire process is coupled to secretion by virtue of the fact that ExsE is a secreted substrate of the T3SS. Changes in the intracellular concentration of ExsE are thought to govern formation of the ExsC-ExsE, ExsC-ExsD, and ExsD-ExsA complexes. Whereas formation of the ExsC-ExsE complex allows ExsD to bind ExsA and transcription of the T3SS is repressed, formation of the ExsC-ExsD complex sequesters ExsD from ExsA and transcription of the T3SS is induced. In this study, we characterized the self-association states of ExsC, ExsD, and ExsE and the binding interactions of ExsC with ExsE and ExsD. ExsC exists as a homodimer and binds one molecule of ExsE substrate. Dimeric ExsC also interacts directly with ExsD to form a heterotetrameric complex. The difference in binding affinities between the ExsC-ExsE (K(d) 1 nm) and ExsC-ExsD (K(d) 18 nm) complexes supports a model in which ExsC preferentially binds cytoplasmic ExsE, resulting in the inhibition of T3SS gene transcription.

Chemical and hormonal determinants of vascular calcification in vitro

Vascular calcification is a complex process that is dependent not only on the physicochemical effects of Ca, PO(4), and pH, but also on smooth muscle factors that may be regulated by these ions as well as by 1,25-dihydroxyvitamin D(3) (calcitriol) and parathyroid hormone (PTH). These minerals and hormones were tested in a model of medial calcification in rat aorta maintained in culture for 9 days. Calcification was quantitated as incorporation of (45)Ca, alkaline phosphatase activity was measured in aortic homogenates, and osteopontin production was measured from immunoblots of culture medium. At 1.8 mM Ca (1.46 mM free), calcification occurred at or above 2.8 mM PO(4). At 3.8 mM PO(4), calcification occurred at or above 1.10 mM free [Ca]. At a constant [Ca] x [PO(4)], calcification varied directly with [Ca] and inversely with [PO(4)]. Calcification was directly related to pH between 7.19 and 7.50 but not altered by PTH or calcitriol. Alkaline phosphatase activity and osteopontin production were increased by Ca, PO(4), calcitriol, and PTH. We conclude that calcification of rat aorta in vitro requires elevation of both [Ca] and [PO(4)], and that [Ca] rather than [PO(4)] or the product of the two is the dominant determinant. The induction of alkaline phosphatase and osteopontin indicates that Ca and PO(4) have effects in addition to simple physicochemical actions. Although PTH and calcitriol did not increase calcification in vivo, they have effects on smooth muscle that could influence calcification in vivo. Calcification is enhanced by alkalinity within the range produced during hemodialysis.

Crystal Structure of the Cytoplasmic Domain of the Chloride Channel ClC-0

Ion channels are frequently organized in a modular fashion and consist of a membrane-embedded pore domain and a soluble regulatory domain. A similar organization is found for the ClC family of Cl- channels and transporters. Here, we describe the crystal structure of the cytoplasmic domain of ClC-0, the voltage-dependent Cl- channel from T. marmorata. The structure contains a folded core of two tightly interacting cystathionine beta-synthetase (CBS) subdomains. The two subdomains are connected by a 96 residue mobile linker that is disordered in the crystals. As revealed by analytical ultracentrifugation, the domains form dimers, thereby most likely extending the 2-fold symmetry of the transmembrane pore. The structure provides insight into the organization of the cytoplasmic domains within the ClC family and establishes a framework for guiding future investigations on regulatory mechanisms.

The thioredoxin system from Streptomyces coelicolor

This paper describes the cloning, purification and characterization of thioredoxin (TrxA) and thioredoxin reductase (TrxR) from bacterial strain Streptomyces coelicolor . The genes of S. coelicolor encoding TrxA and TrxR were amplified by polymerase chain reaction, inserted into pET expression vector and used to overexpress these proteins in Escherichia coli . TrxA and TrxR were produced as the hexahistidine fusion proteins and were recovered from the cytoplasm as the soluble proteins. The activity of the purified recombinant proteins was demonstrated. The activity of TrxA was shown by efficient reduction of insulin and activity of NADPH-dependent TrxR was revealed by catalyses of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) reduction reaction. The reduction reaction was fully dependent upon the presence of TrxA as intermediate electron carrier with the pH optimum 7.5 and the temperature optimum 29 degrees C. Km value of TrxR for TrxA was 0.217 +/- 0.02 microM.

Synthesis of an Artificial Cell Surface Receptor that Enables Oligohistidine Affinity Tags to Function as Metal-Dependent Cell-Penetrating Peptides

Cell-penetrating peptides and proteins (CPPs) are important tools for the delivery of impermeable molecules into living mammalian cells. To enable these cells to internalize proteins fused to common oligohistidine affinity tags, we synthesized an artificial cell surface receptor comprising an N-alkyl derivative of 3beta-cholesterylamine linked to the metal chelator nitrilotriacetic acid (NTA). This synthetic receptor inserts into cellular plasma membranes, projects NTA headgroups from the cell surface, and rapidly cycles between the plasma membrane and intracellular endosomes. Jurkat lymphocytes treated with the synthetic receptor (10 microM) for 1 h displayed approximately 8,400,000 [corrected]NTA groups on the cell surface. Subsequent addition of the green fluorescent protein AcGFP fused to hexahistidine or decahistidine peptides (3 microM) and Ni(OAc)(2) (100 microM) enhanced the endocytosis of AcGFP by 150-fold (hexahistidine fusion protein) or 600-fold (decahistidine fusion protein) within 4 h at 37 degrees C. No adverse effects on cellular proliferation or morphology were observed under these conditions. By enabling common oligohistidine affinity tags to function as cell-penetrating peptides, this metal-chelating cell surface receptor provides a useful tool for studies of cellular biology [corrected]

Expression, purification, and characterization of the 4 zinc finger region of human tumor suppressor WT1

Wilm’s Tumor gene 1 (WT1) encodes a zinc finger protein with four distinct splice isoforms. WT1 has a critical role in genesis of various cancer types both at the DNA/RNA and the protein level. The zinc-finger DNA-binding capacity of the protein is located in the C-terminal domain. Two recombinant proteins, 6HIS-ZN− wt1 and 6HIS-ZN+ wt1, corresponding to two alternative splice variants of the C-terminal regions of human WT1 (−KTS) and WT1 (+KTS), respectively, were over-expressed with hexa-histidine fusion tags in inclusion bodies in Escherichia coli for crystallization studies. A combination of Ni 2+–NTA affinity and size-exclusion chromatography was applied for purification of the proteins in denaturing conditions. The effects of various buffers, salts and other additives were scrutinized in a systematic screening to establish the optimal conditions for solubility and refolding of the recombinant WT1 proteins. Circular dichroism analysis revealed the expected ββα content for the refolded proteins, with a notable degradation of the α-helical segment in the DNA-free state. Electrophoretic mobility shift assay with double-stranded DNA containing the double Egr1 consensus site 5′-GCG-TGG-GCG-3′ confirmed that 6HIS-ZN− wt1 has higher DNA binding affinity than 6HIS-ZN+ wt1.

Molecular and Biochemical Characterization of the xlnD -Encoded 3-Hydroxybenzoate 6-Hydroxylase Involved in the Degradation of 2,5-Xylenol via the Gentisate Pathway in Pseudomonas alcaligenes NCIMB 9867

The xlnD gene from Pseudomonas alcaligenes NCIMB 9867 (strain P25X) was shown to encode 3-hydroxybenzoate 6-hydroxylase I, the enzyme that catalyzes the NADH-dependent conversion of 3-hydroxybenzoate to gentisate. Active recombinant XlnD was purified as a hexahistidine fusion protein from Escherichia coli, had an estimated molecular mass of 130 kDa, and is probably a trimeric protein with a subunit mass of 43 kDa. This is in contrast to the monomeric nature of the few 3-hydroxybenzoate 6-hydroxylases that have been characterized thus far. Like other 3-hydroxybenzoate 6-hydroxylases, XlnD could utilize either NADH or NADPH as the electron donor. P25X harbors a second 3-hydroxybenzoate 6-hydroxylase II that was strictly inducible by specific aromatic substrates. However, the degradation of 2,5-xylenol and 3,5-xylenol in strain P25X was found to be dependent on the xlnD-encoded 6-hydroxylase I and not the second, strictly inducible 6-hydroxylase II.

Cloning and characterization of an Arabidopsis thaliana Nudix hydrolase homologous to the mammalian GFG protein

In a search for a plant antimutator MutT protein, an Arabidopsis thaliana Nudix hydrolase with homology to the mammalian GFG protein was expressed as a hexahistidine fusion polypeptide in Escherichia coli and purified to homogeneity. Unlike the GFG protein, the A. thaliana homolog could not complement the mutT mutation in a MutT-deficient E. coli strain nor was it able to hydrolyze 8-oxo-dGTP, the main substrate of the MutT protein. Instead the recombinant protein hydrolyzed a variety of nucleoside diphosphate derivatives showing a preference for ADP-ribose, with K m and k cat values of 1.2 mM and 2.7 s −1 respectively. The products of ADP-ribose hydrolysis were AMP and ribose-5-phosphate. The optimal activity was at alkaline pH (8.5) with Mg 2+ (5 mM) ions as the cofactor. The protein exists as a dimmer in solution.

Salicylate Biosynthesis: Overexpression, Purification, and Characterization of Irp9, a Bifunctional Salicylate Synthase fromYersinia enterocolitica

In some bacteria, salicylate is synthesized using the enzymes isochorismate synthase and isochorismate pyruvate lyase. In contrast, gene inactivation and complementation experiments with Yersinia enterocolitica suggest the synthesis of salicylate in the biosynthesis of the siderophore yersiniabactin involves a single protein, Irp9, which converts chorismate directly into salicylate. In the present study, Irp9 was for the first time heterologously expressed in Escherichia coli as a hexahistidine fusion protein, purified to near homogeneity, and characterized biochemically. The recombinant protein was found to be a dimer, each subunit of which has a molecular mass of 50 kDa. Enzyme assays, reverse-phase high-pressure liquid chromatography and 1H nuclear magnetic resonance (NMR) spectroscopic analyses confirmed that Irp9 is a salicylate synthase and converts chorismate to salicylate with a K(m) for chorismate of 4.2 microM and a k(cat) of 8 min(-1). The reaction was shown to proceed through the intermediate isochorismate, which was detected directly using 1H NMR spectroscopy.