Reduction of OsMPK6 activity by a R89K mutation induces cell death and bacterial blight resistance in rice.

KEY MESSAGE: The R89 is essential for the kinase activity of OsMPK6 which negatively regulates cell death and defense response in rice. Mitogen-activated protein kinase cascade plays critical roles in various vital activities, including the plant immune response, but the mechanisms remain elusive. Here, we identified and characterized a rice lesion mimic mutant osmpk6 which displayed hypersensitive response-like lesions in company with cell death and hydrogen peroxide hyperaccumulation. Map-based cloning and complementation demonstrated that a G702A single-base substitution in the second exon of OsMPK6 led to the lesion mimic phenotype of the osmpk6 mutant. OsMPK6 encodes a cytoplasm and nucleus-targeted mitogen-activated protein kinase and is expressed in the various organs. Compared with wild type, the osmpk6 mutant exhibited high resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), likely due to the increased ROS production induced by flg22 and chitin and up-regulated expression of genes involved in pathogenesis, as well as activation of SA and JA signaling pathways after inoculation. By contrast, the OsMPK6-overexpression line (OE-1) was found to be susceptible to the bacterial pathogens, indicating that OsMPK6 negatively regulated Xoo resistance. Furthermore, the G702A single-base substitution caused a R89K mutation at both polypeptide substrate-binding site and active site of OsMPK6, and kinase activity assay revealed that the R89K mutation led to reduction of OsMPK6 activity, suggesting that the R89 is essential for the function of OsMPK6. Our findings provide insight into a vital role of the R89 of OsMPK6 in regulating cell death and defense response in rice.

Site-Selective Functionalization of Nanobodies Using Intein-Mediated Protein Ligation for Innovative Bioconjugation.

An expression strategy is presented in order to produce nanobodies modified with a clickable alkyne functionality at their C-terminus via the intein-mediated protein ligation (IPL) technique. The protocol focuses on the cytoplasmic expression and extraction of a nanobody-intein-chitin binding domain (CBD) fusion protein in E. coli SHuffle® T7 cells, in the commonly used Luria-Bertani (LB) medium. The combination of these factors results in a high yield and nearly complete alkynation of the nanobody at its C-terminus via IPL. The resulting alkynated nanobodies retain excellent binding capacity toward the nanobody targeted antigen. The presented protocol benefits from time- and cost-effectiveness and allows for a feasible upscaling of functionalized (here alkynated) nanobodies. The production of high quantities of site-specifically modified nanobodies paves the way to (1) novel biosurface applications that demand for homogeneously oriented nanobodies having their active site fully accessible for target (e.g., biomarker) binding, and (2) innovative applications such as localized drug delivery and image guided surgery by covalent "click" chemistry coupling of these alkynated nanobodies to a multitude of azide-containing counterparts as there are drug containing polymers and contrast labeling agents.

Knockdown of the chitin-binding protein family gene CaChiIV1 increased sensitivity to Phytophthora capsici and drought stress in pepper plants.

Phytophthora capsici has been the most destructive pathogen of pepper plants (Capsicum annuum L.), possessing the ability to quickly overcome the host defense system. In this context, the chitin-binding protein (CBP) family member CaChiIV1 regulates the response to P. capsici and abiotic stresses. The relevance of functional characterization and regulation of CaChiIV1 has not been explored in horticultural crops, especially pepper plants. The target gene (CaChiIV1) was isolated from pepper plants and cloned; the encoded protein carries a chitin-binding domain (CBD) that is rich in cysteine residues and has a hinge region with an abundance of proline and glycine residues. Additionally, the conserved regions in the promoter have a remarkable motif, "TTGACC". The expression of CaChiIV1 was markedly regulated by methyl-jasmonate (MeJA), hydrogen peroxide (H2O2), melatonin, mannitol and P. capsici (PC and HX-9) infection. Knockdown of CaChiIV1 in pepper plants increased sensitivity to P. capsici (PC strain). Higher malondialdehyde (MDA) content and relative electrolyte leakage (REL) but lower antioxidant enzyme activities, chlorophyll content, root activity, and proline content were observed in CaChiIV1-silenced plants than in control plants. In conclusion, CaChiIV1-silenced pepper plants displayed increased susceptibility to P. capsici infection due to changes in expression of defense-related genes, thus showing its coregulation affect in particular conditions. Furthermore, antioxidant enzymes and proline content were largely diminished in CaChiIV1-silenced plants. Therefore, this evidence suggests that the CaChiIV1 gene plays a prominent role in the defense mechanism of pepper plants against P. capsici infection. In the future, the potential role of the CaChiIV1 gene in defense regulatory pathways and its coregulation with other pathogen-related genes should be identified.

Conserved function of the matriptase-prostasin proteolytic cascade during epithelial morphogenesis.

Extracellular matrix (ECM) assembly and remodelling is critical during development and organ morphogenesis. Dysregulation of ECM is implicated in many pathogenic conditions, including cancer. The type II transmembrane serine protease matriptase and the serine protease prostasin are key factors in a proteolytic cascade that regulates epithelial ECM differentiation during development in vertebrates. Here, we show by rescue experiments that the Drosophila proteases Notopleural (Np) and Tracheal-prostasin (Tpr) are functional homologues of matriptase and prostasin, respectively. Np mediates morphogenesis and remodelling of apical ECM during tracheal system development and is essential for maintenance of the transepithelial barrier function. Both Np and Tpr degrade the zona pellucida-domain (ZP-domain) protein Dumpy, a component of the transient tracheal apical ECM. Furthermore, we demonstrate that Tpr zymogen and the ZP domain of the ECM protein Piopio are cleaved by Np and matriptase in vitro. Our data indicate that the evolutionarily conserved ZP domain, present in many ECM proteins of vertebrates and invertebrates, is a novel target of the conserved matriptase-prostasin proteolytic cascade.

Long-Chain Polyprenols Promote Spore Wall Formation in Saccharomyces cerevisiae.

Dolichols are isoprenoid lipids of varying length that act as sugar carriers in glycosylation reactions in the endoplasmic reticulum. In Saccharomyces cerevisiae, there are two cis-prenyltransferases that synthesize polyprenol-an essential precursor to dolichol. These enzymes are heterodimers composed of Nus1 and either Rer2 or Srt1. Rer2-Nus1 and Srt1-Nus1 can both generate dolichol in vegetative cells, but srt1∆ cells grow normally while rer2∆ grows very slowly, indicating that Rer2-Nus1 is the primary enzyme used in mitotically dividing cells. In contrast, SRT1 performs an important function in sporulating cells, where the haploid genomes created by meiosis are packaged into spores. The spore wall is a multilaminar structure and SRT1 is required for the generation of the outer chitosan and dityrosine layers of the spore wall. Srt1 specifically localizes to lipid droplets associated with spore walls, and, during sporulation there is an SRT1-dependent increase in long-chain polyprenols and dolichols in these lipid droplets. Synthesis of chitin by Chs3, the chitin synthase responsible for chitosan layer formation, is dependent on the cis-prenyltransferase activity of Srt1, indicating that polyprenols are necessary to coordinate assembly of the spore wall layers. This work shows that a developmentally regulated cis-prenyltransferase can produce polyprenols that function in cellular processes besides protein glycosylation.

A broad pH range and processive chitinase from a metagenome library

Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.

Mo-CBP3, an antifungal chitin-binding protein from Moringa oleifera seeds, is a member of the 2S albumin family.

Mo-CBP3 is a chitin-binding protein from M. oleifera seeds that inhibits the germination and mycelial growth of phytopathogenic fungi. This protein is highly thermostable and resistant to pH changes, and therefore may be useful in the development of new antifungal drugs. However, the relationship of MoCBP3 with the known families of carbohydrate-binding domains has not been established. In the present study, full-length cDNAs encoding 4 isoforms of Mo-CBP3 (Mo-CBP3-1, Mo-CBP3-2, Mo-CBP3-3 and Mo-CBP3-4) were cloned from developing seeds. The polypeptides encoded by the Mo-CBP3 cDNAs were predicted to contain 160 (Mo-CBP3-3) and 163 amino acid residues (Mo-CBP3-1, Mo-CBP3-2 and Mo-CBP3-4) with a signal peptide of 20-residues at the N-terminal region. A comparative analysis of the deduced amino acid sequences revealed that Mo-CBP3 is a typical member of the 2S albumin family, as shown by the presence of an eight-cysteine motif, which is a characteristic feature of the prolamin superfamily. Furthermore, mass spectrometry analysis demonstrated that Mo-CBP3 is a mixture of isoforms that correspond to different mRNA products. The identification of Mo-CBP3 as a genuine member of the 2S albumin family reinforces the hypothesis that these seed storage proteins are involved in plant defense. Moreover, the chitin-binding ability of Mo-CBP3 reveals a novel functionality for a typical 2S albumin.

Obstructor A organizes matrix assembly at the apical cell surface to promote enzymatic cuticle maturation in Drosophila.

Assembly and maturation of the apical extracellular matrix (aECM) is crucial for protecting organisms, but underlying molecular mechanisms remain poorly understood. Epidermal cells secrete proteins and enzymes that assemble at the apical cell surface to provide epithelial integrity and stability during developmental growth and upon tissue damage. We analyzed molecular mechanisms of aECM assembly and identified the conserved chitin-binding protein Obst-A (Obstructor A) as an essential regulator. We show in Drosophila that Obst-A is required to coordinate protein and chitin matrix packaging at the apical cell surface during development. Secreted by epidermal cells, the Obst-A protein is specifically enriched in the apical assembly zone where matrix components are packaged into their highly ordered architecture. In obst-A null mutant larvae, the assembly zone is strongly diminished, resulting in severe disturbance of matrix scaffold organization and impaired aECM integrity. Furthermore, enzymes that support aECM stability are mislocalized. As a biological consequence, cuticle architecture, integrity, and function are disturbed in obst-A mutants, finally resulting in immediate lethality upon wounding. Our studies identify a new core organizing center, the assembly zone that controls aECM assembly at the apical cell surface. We propose a genetically conserved molecular mechanism by which Obst-A forms a matrix scaffold to coordinate trafficking and localization of proteins and enzymes in the newly deposited aECM. This mechanism is essential for maturation and stabilization of the aECM in a growing and remodeling epithelial tissue as an outermost barrier.

Chitin is endogenously produced in vertebrates.

Chitin, a biopolymer of N-acetylglucosamine, is abundant in invertebrates and fungi and is an important structural molecule [1, 2]. There has been a longstanding belief that vertebrates do not produce chitin; however, we have obtained compelling evidence to the contrary. Chitin synthase genes are present in numerous fishes and amphibians, and chitin is localized in situ to the lumen of the developing zebrafish gut, in epithelial cells of fish scales, and in at least three different cell types in larval salamander appendages. Chitin synthase gene knockdowns and various histochemical experiments in zebrafish further authenticated our results. Finally, a polysaccharide was extracted from scales of salmon that exhibited all the chemical hallmarks of chitin. Our data and analyses demonstrate the existence of endogenous chitin in vertebrates and suggest that it serves multiple roles in vertebrate biology.

High prevalence of chitotriosidase deficiency in Peruvian Amerindians exposed to chitin-bearing food and enteroparasites.

The human genome encodes a gene for an enzymatically active chitinase (CHIT1) located in a single copy on Chromosome 1, which is highly expressed by activated macrophages and in other cells of the innate immune response. Several dysfunctional mutations are known in CHIT1, including a 24-bp duplication in Exon 10 causing catalytic deficiency. This duplication is a common variant conserved in many human populations, except in West and South Africans. Thus it has been proposed that human migration out of Africa and the consequent reduction of exposure to chitin from environmental factors may have enabled the conservation of dysfunctional mutations in human chitinases. Our data obtained from 85 indigenous Amerindians from Peru, representative of populations characterized by high prevalence of chitin-bearing enteroparasites and intense entomophagy, reveal a very high frequency of the 24-bp duplication (47.06%), and of other single nucleotide polymorphisms which are known to partially affect enzymatic activity (G102S: 42.7% and A442G/V: 25.5%). Our finding is in line with a founder effect, but appears to confute our previous hypothesis of a protective role against parasite infection and sustains the discussion on the redundancy of chitinolytic function.

Ectopic expression of the male BmDSX affects formation of the chitin plate in female Bombyx mori.

Mating structures are involved in successful copulation, intromission, and/or insemination. These structures enable tight coupling between external genitalia of two sexes. During Bombyx mori copulation, the double harpagones in the external genitalia of males clasp the female chitin plate, which is derived from the larval eighth abdominal segment; abnormal development of the female chitin plate affects copulation. We report that ERK phosphorylation (p-ERK) and expression of Abdominal-B (Abd-B) in the posterior abdomen of the female adult is lower than in the male. Ectopic expression of the male-specific spliced form of B. mori doublesex (Bmdsx(M)) in females, however, up-regulates Abd-B and spitz (spi) expression, increasing EGFR signaling activity, and thus forming an abnormal chitin plate and reduced female copulation. These findings indicate that Bmdsx affects the development of the eighth abdominal segment by regulating the activity of EGFR signaling and the expression of Abd-B, resulting in an extra eighth abdominal segment (A8) in males versus the loss of this segment in adult females.

Cooperative degradation of chitin by extracellular and cell surface-expressed chitinases from Paenibacillus sp. strain FPU-7.

Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine.

Functional differentiation of chitin synthases in Yarrowia lipolytica.

In this study, we identified seven chitin synthase-encoding genes in the genome of the dimorphic yeast Yarrowia lipolytica. Three encoded chitin synthases with myosin motor-like domains at their N-termini, and we designated these CSM1 to CSM3, whereas four were identified as CHS1 to CHS4. To investigate the functions of these seven genes, we constructed and characterized their deletion mutants. The chs2Δ mutant formed chained cells in which daughter cells were connected with mother cells and had abnormally thick septa at the bud neck. The chs4Δ mutant showed remarkably reduced chitin content in its cell wall. The chs2Δ, csm1Δ, and csm2Δ mutants were found to be highly sensitive to chitin binding dyes, calcofluor white (CFW) and Congo red, whereas the chs4Δ mutant was resistant to CFW. These results suggest that Chs2 and Chs4 play major roles in septum formation and cell wall chitin synthesis respectively, whereas Csm1 and Csm2 are involved in the maintenance of cell wall architecture and/or cell wall integrity. The populations of filamentous cells, a type of cell population that are defined by the lengths of the cellular long and short axes, decreased in the chs3Δ mutant, suggesting that Chs3 is involved in cellular morphogenesis.

Gene expression modulation by paraquat-induced oxidative stress conditions in Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a thermodimorphic fungus associated with paracoccidioidomycosis (PCM), the most common systemic mycosis in Latin America. The infection is initiated by inhalation of environmentally dispersed conidia produced by the saprophytic phase of the fungus. In the lungs, P. brasiliensis assumes the parasitic yeast form and must cope with the adverse conditions imposed by cells of the host immune system, which includes a harsh environment, highly concentrated in reactive oxygen species (ROS). In this work, we used the ROS-generating agent paraquat to experimentally simulate oxidative stress conditions in order to evaluate the stress-induced modulation of gene expression in cultured P. brasiliensis yeast cells, using a microarray hybridization approach. The large-scale evaluation inherent to microarray-based analyses identified 2070 genes differentially transcribed in response to paraquat exposure, allowing an integrated visualization of the major metabolic changes that constitute the systemic defense mechanism used by the fungus to overcome the deleterious effects of ROS. These include overexpression of detoxifying agents, as well as of molecular scavengers and genes involved in maintenance of the intracellular redox potential. Particularly noteworthy was to verify that the oxidative stress resistance mechanism of P. brasiliensis also involves coordinated overexpression of a series of genes responsible for chitin-biosynthesis, suggesting that this pathway may constitute a specific regulon. Further analyses aiming at confirming and understanding the mechanisms that control such regulon may provide interesting new targets for chemotherapeutic approaches against P. brasiliensis and other pathogenic fungi.

Retroactive maintains cuticle integrity by promoting the trafficking of Knickkopf into the procuticle of Tribolium castaneum.

Molting, or the replacement of the old exoskeleton with a new cuticle, is a complex developmental process that all insects must undergo to allow unhindered growth and development. Prior to each molt, the developing new cuticle must resist the actions of potent chitinolytic enzymes that degrade the overlying old cuticle. We recently disproved the classical dogma that a physical barrier prevents chitinases from accessing the new cuticle and showed that the chitin-binding protein Knickkopf (Knk) protects the new cuticle from degradation. Here we demonstrate that, in Tribolium castaneum, the protein Retroactive (TcRtv) is an essential mediator of this protective effect of Knk. TcRtv localizes within epidermal cells and specifically confers protection to the new cuticle against chitinases by facilitating the trafficking of TcKnk into the procuticle. Down-regulation of TcRtv resulted in entrapment of TcKnk within the epidermal cells and caused molting defects and lethality in all stages of insect growth, consistent with the loss of TcKnk function. Given the ubiquity of Rtv and Knk orthologs in arthropods, we propose that this mechanism of new cuticle protection is conserved throughout the phylum.

Chitin-binding proteins of Artemia diapause cysts participate in formation of the embryonic cuticle layer of cyst shells.

The brine shrimp Artemia reproduces either ovoviviparously, producing free-swimming nauplii, or oviparously, producing encysted embryos (diapause cysts) able to cope with harsh and complex habitats. When the cysts enter diapause they are encased in a complex external shell that protects them from certain extreme environments. The genomic comparison of oviparous and ovoviviparous ovisacs has been described previously. We isolated three significantly up-regulated genes in oviparous oocytes and identified them as Arp-CBP (Artemia parthenogenetica chitin-binding protein) genes. Quantitative real-time PCR indicated that the expression of Arp-CBP genes gradually increases during diapause cyst formation and significant mRNA accumulation occurs during the ovisac stage of oviparous development. Moreover, in situ hybridization results demonstrated that Arp-CBP mRNAs are expressed in the embryo. Interestingly, the results of immune electron microscopy showed that all three Arp-CBPs are distributed throughout the cellular ECL (embryonic cuticle layer) of the cyst shell. Furthermore, knockdown of Arp-CBP by RNA interference resulted in marked changes in the composition of the embryonic cuticular layer. The fibrous layer of the cyst shell adopted a loose conformation and the inner and outer cuticular membranes exhibited marked irregularities when Arp-CBP expression was suppressed. Finally, an in vitro recombinant protein-binding assay showed that all three Arp-CBPs have carbohydrate-binding activities. These findings provide significant insight into the mechanisms by which the ECL of Artemia cyst shell is formed, and demonstrate that Arp-CBPs are involved in construction of the fibrous lattice and are required for formation of the ECL of the cyst shell.

Gene families of cuticular proteins analogous to peritrophins (CPAPs) in Tribolium castaneum have diverse functions.

The functional characterization of an entire class of 17 genes from the red flour beetle, Tribolium castaneum, which encode two families of Cuticular Proteins Analogous to Peritrophins (CPAPs) has been carried out. CPAP genes in T. castaneum are expressed exclusively in cuticle-forming tissues and have been classified into two families, CPAP1 and CPAP3, based on whether the proteins contain either one (CPAP1), or three copies (CPAP3) of the chitin-binding domain, ChtBD2, with its six characteristically spaced cysteine residues. Individual members of the TcCPAP1 and TcCPAP3 gene families have distinct developmental patterns of expression. Many of these proteins serve essential and non-redundant functions in maintaining the structural integrity of the cuticle in different parts of the insect anatomy. Three genes of the TcCPAP1 family and five genes of the TcCPAP3 family are essential for insect development, molting, cuticle integrity, proper locomotion or fecundity. RNA interference (RNAi) targeting TcCPAP1-C, TcCPAP1-H, TcCPAP1-J or TcCPAP3-C transcripts resulted in death at the pharate adult stage of development. RNAi for TcCPAP3-A1, TcCPAP3-B, TcCPAP3-D1 or TcCPAP3-D2 genes resulted in different developmental defects, including adult/embryonic mortality, abnormal elytra or hindwings, or an abnormal 'stiff-jointed' gait. These results provide experimental support for specialization in the functions of CPAP proteins in T. castaneum and a biological rationale for the conservation of CPAP orthologs in other orders of insects. This is the first comprehensive functional analysis of an entire class of cuticular proteins with one or more ChtBD2 domains in any insect species.

No jacket required--new fungal lineage defies dress code: recently described zoosporic fungi lack a cell wall during trophic phase.

Analyses of environmental DNAs have provided tantalizing evidence for "rozellida" or "cryptomycota", a clade of mostly undescribed and deeply diverging aquatic fungi. Here, we put cryptomycota into perspective through consideration of Rozella, the only clade member growing in culture. This is timely on account of the publication in Nature of the first images of uncultured cryptomycota from environmental filtrates, where molecular probes revealed non-motile cyst-like structures and motile spores, all lacking typical fungal chitinous cell walls. Current studies of Rozella can complement these fragmentary observations from environmental samples. Rozella has a fungal-specific chitin synthase and its resting sporangia have walls that appear to contain chitin. Cryptomycota, including Rozella, lack a cell wall when absorbing food but like some other fungi, they may have lost their "dinner jacket" through convergence. Rather than evolutionary intermediates, the cryptomycota may be strange, divergent fungi that evolved from an ancestor with a nearly complete suite of classical fungal-specific characters.

Microbial synthesis of hyaluronan and chitin: New approaches.

Hyaluronan (HA) is an important structural element in the vitreous humor of the eye, synovial fluid, and skin of vertebrates. Moreover, HA interacts with proteins such as CD44, RHAMM, and fibrinogen, thereby influencing many natural processes such as angiogenesis, cancer, cell motility, wound healing, and cell adhesion. Reflecting such a variety of functions, HA has attracted attention from a wide range of application fields such as medicine (including surgery), cosmetics, and health foods. Traditionally HA was extracted from rooster combs, but nowadays is produced by the fermentation of streptococci. At present, quality issues such as purity and molecular weight distribution, rather than quantity, have been the focus of strain and process development in HA production. To meet ever-increasing public demand, novel systems that can yield sufficient amounts of high-quality of HA and related materials are required.

Zinc ion effects on individual Ssp DnaE intein splicing steps: regulating pathway progression.

Use of the naturally split, self-splicing Synechocystis sp. PCC6803 DnaE intein permits separate purification of the N- and C-terminal intein domains. Otherwise spontaneous intein-mediated reactions can therefore be controlled in vitro, allowing detailed study of intein kinetics. Incubation of the Ssp DnaE intein with ZnCl(2) inhibited trans splicing, hydrolysis-mediated N-terminal trans cleavage, and C-terminal trans cleavage reactions. Maximum inhibition of the splicing reaction was achieved at equal molar concentrations of ZnCl(2) and intein domains, suggesting a 1:1 metal ion:intein binding stoichiometry. Mutation of the (+)1 cysteine residue to valine (C(+)1V) alleviated the inhibitory effects of ZnCl(2). Valine substitution in the absence of ZnCl(2) blocked trans splicing and decreased C-terminal cleavage kinetics in a manner similar to that of the native (+)1 cysteine in the presence of ZnCl(2). These data are consistent with Zn(2+)-mediated inhibition of the Ssp DnaE intein via chelation of the (+)1 cysteine residue. N-Terminal trans cleavage can occur via both spontaneous hydrolysis and nucleophilic (e.g., DTT) attack. Comparative examination of N-terminal cleavage rates using amino acid substitution (C(+)1V) and Zn(2+)-mediated inhibition permitted the maximum contribution of hydrolysis to overall N-terminal cleavage kinetics to be determined. Stable intermediates consisting of the associated intein domains were detected by PAGE and provided evidence of a rapid C-terminal cleavage step. Acute control of the C-terminal reaction was achieved by the rapid reversal of Zn(2+)-mediated inhibition by EDTA. By inhibiting both the splicing pathway and spontaneous hydrolysis with Zn(2+), reactants can be diverted from the trans splicing to the trans cleavage pathway where DTT and EDTA can regulate N- and C-terminal cleavage, respectively.