Processing of a plant peptide hormone precursor facilitated by posttranslational tyrosine sulfation.

Most peptide hormones and growth factors are matured from larger inactive precursor proteins by proteolytic processing and further posttranslational modification. Whether or how posttranslational modifications contribute to peptide bioactivity is still largely unknown. We address this question here for TWS1 (Twisted Seed 1), a peptide regulator of embryonic cuticle formation in Arabidopsis thaliana. Using synthetic peptides encompassing the N- and C-terminal processing sites and the recombinant TWS1 precursor as substrates, we show that the precursor is cleaved by the subtilase SBT1.8 at both the N and the C termini of TWS1. Recognition and correct processing at the N-terminal site depended on sulfation of an adjacent tyrosine residue. Arginine 302 of SBT1.8 was found to be required for sulfotyrosine binding and for accurate processing of the TWS1 precursor. The data reveal a critical role for posttranslational modification, here tyrosine sulfation of a plant peptide hormone precursor, in mediating processing specificity and peptide maturation.

Inhibitory activity of bacterial lipopeptides against Fusarium oxysporum f.sp. Strigae.

This study investigated the influence of bacterial cyclic lipopeptides (LP; surfactins, iturins, fengycins) on microbial interactions. The objective was to investigate whether the presence of bacteria inhibits fungal growth and whether this inhibition is due to the release of bacterial metabolites, particularly LP. Selected endophytic bacterial strains with known plant-growth promoting potential were cultured in the presence of Fusarium oxysporum f.sp. strigae (Fos), which was applied as model fungal organism. The extracellular metabolome of tested bacteria, with a focus on LP, was characterized, and the inhibitory effect of bacterial LP on fungal growth was investigated. The results showed that Bacillus velezensis GB03 and FZB42, as well as B. subtilis BSn5 exhibited the strongest antagonism against Fos. Paraburkholderia phytofirmans PsJN, on the other hand, tended to have a slight, though non-significant growth promotion effect. Crude LP from strains GB03 and FZB42 had the strongest inhibitory effect on Fos, with a significant inhibition of spore germination and damage of the hyphal structure. Liquid chromatography tandem mass spectrometry revealed the production of several variants of iturin, fengycin, and surfactin LP families from strains GB03, FZB42, and BSn5, with varying intensity. Using plate cultures, bacillomycin D fractions were detected in higher abundance in strains GB03, FZB42, and BSn5 in the presence of Fos. Additionally, the presence of Fos in dual plate culture triggered an increase in bacillomycin D production from the Bacillus strains. The study demonstrated the potent antagonistic effect of certain Bacillus strains (i.e., GB03, FZB42, BSn5) on Fos development. Our findings emphasize the crucial role of microbial interactions in shaping the co-existence of microbial assemblages.

A novel, robust peptidyl-lys metalloendopeptidase from Trametes coccinea recombinantly expressed in Komagataella phaffii.

A novel peptidyl-lys metalloendopeptidase (Tc-LysN) from Tramates coccinea was recombinantly expressed in Komagataella phaffii using the native pro-protein sequence. The peptidase was secreted into the culture broth as zymogen (~38 kDa) and mature enzyme (~19.8 kDa) simultaneously. The mature Tc-LysN was purified to homogeneity with a single step anion-exchange chromatography at pH 7.2. N-terminal sequencing using TMTpro Zero and mass spectrometry of the mature Tc-LysN indicated that the pro-peptide was cleaved between the amino acid positions 184 and 185 at the Kex2 cleavage site present in the native pro-protein sequence. The pH optimum of Tc-LysN was determined to be 5.0 while it maintained ≥60% activity between pH values 4.5-7.5 and ≥30% activity between pH values 8.5-10.0, indicating its broad applicability. The temperature maximum of Tc-LysN was determined to be 60 °C. After 18 h of incubation at 80 °C, Tc-LysN still retained ~20% activity. Organic solvents such as methanol and acetonitrile, at concentrations as high as 40% (v/v), were found to enhance Tc-LysN's activity up to ~100% and ~50%, respectively. Tc-LysN's thermostability, ability to withstand up to 8 M urea, tolerance to high concentrations of organic solvents, and an acidic pH optimum make it a viable candidate to be employed in proteomics workflows in which alkaline conditions might pose a challenge. The nano-LC-MS/MS analysis revealed bovine serum albumin (BSA)'s sequence coverage of 84% using Tc-LysN which was comparable to the sequence coverage of 90% by trypsin peptides. KEY POINTS: •A novel LysN from Trametes coccinea (Tc-LysN) was expressed in Komagataella phaffii and purified to homogeneity •Tc-LysN is thermostable, applicable over a broad pH range, and tolerates high concentrations of denaturants •Tc-LysN was successfully applied for protein digestion and mass spectrometry fingerprinting.

14-3-3 and erlin proteins differentially interact with RIPK2 complexes.

The receptor interacting serine/threonine kinase 2 (RIPK2) is essential for signal transduction induced by the pattern recognition receptors NOD1 and NOD2 (referred to collectively as NOD1/2). Upon NOD1/2 activation, RIPK2 forms complexes in the cytoplasm of human cells. Here, we identified the molecular composition of these complexes. Infection with Shigella flexneri to activate NOD1-RIPK2 revealed that RIPK2 formed dynamic interactions with several cellular proteins, including A20 (also known as TNFAIP3), erlin-1, erlin-2 and 14-3-3. Whereas interaction of RIPK2 with 14-3-3 proteins was strongly reduced upon infection with Shigella, erlin-1 and erlin-2 (erlin-1/2) specifically bound to RIPK2 complexes. The interaction of these proteins with RIPK2 was validated using protein binding assays and immunofluorescence staining. Beside bacterial activation of NOD1/2, depletion of the E3 ubiquitin ligase XIAP and treatment with RIPK2 inhibitors also led to the formation of RIPK2 cytosolic complexes. Although erlin-1/2 were recruited to RIPK2 complexes following XIAP inhibition, these proteins did not associate with RIPK2 structures induced by RIPK2 inhibitors. While the specific recruitment of erlin-1/2 to RIPK2 suggests a role in innate immune signaling, the biological response regulated by the erlin-1/2-RIPK2 association remains to be determined.

Mineral and Phytic Acid Content as Well as Phytase Activity in Flours and Breads Made from Different Wheat Species.

Wheat is of high importance for a healthy and sustainable diet for the growing world population, partly due to its high mineral content. However, several minerals are bound in a phytate complex in the grain and unavailable to humans. We performed a series of trials to compare the contents of minerals and phytic acid as well as phytase activity in several varieties from alternative wheat species spelt, emmer and einkorn with common wheat. Additionally, we investigated the potential of recent popular bread making recipes in German bakeries to reduce phytic acid content, and thus increase mineral bioavailability in bread. For all studied ingredients, we found considerable variance both between varieties within a species and across wheat species. For example, whole grain flours, particularly from emmer and einkorn, appear to have higher mineral content than common wheat, but also a higher phytic acid content with similar phytase activity. Bread making recipes had a greater effect on phytic acid content in the final bread than the choice of species for whole grain flour production. Recipes with long yeast proofing or sourdough and the use of whole grain rye flour in a mixed wheat bread minimized the phytic acid content in the bread. Consequently, optimizing food to better nourish a growing world requires close collaboration between research organizations and practical stakeholders ensuring a streamlined sustainable process from farm to fork.

Life at the periphery: what makes CHO cells survival talents.

The production of biopharmaceuticals relies on robust cell systems that can produce recombinant proteins at high levels and grow and survive in the stressful bioprocess environment. Chinese hamster ovary cells (CHO) as the main production hosts offer a variety of advantages including robust growth and survival in a bioprocess environment. Cell surface proteins are of special interest for the understanding of how CHO cells react to their environment while maintaining growth and survival phenotypes, since they enable cellular reactions to external stimuli and potentially initiate signaling pathways. To provide deeper insight into functions of this special cell surface sub-proteome, pathway enrichment analysis of the determined CHO surfaceome was conducted. Enrichment of growth/ survival-pathways such as the phosphoinositide-3-kinase (PI3K)-protein kinase B (AKT), mitogen-activated protein kinase (MAPK), Janus kinase/signal transducers and activators of transcription (JAK-STAT), and RAP1 pathways were observed, offering novel insights into how cell surface receptors and ligand-mediated signaling enable the cells to grow and survive in a bioprocess environment. When supplementing surfaceome data with RNA expression data, several growth/survival receptors were shown to be co-expressed with their respective ligands and thus suggesting self-induction mechanisms, while other receptors or ligands were not detectable. As data about the presence of surface receptors and their associated expressed ligands may serve as base for future studies, further pathway characterization will enable the implementation of optimization strategies to further enhance cellular growth and survival behavior. KEY POINTS: • PI3K/AKT, MAPK, JAK-STAT, and RAP1 pathway receptors are enriched on the CHO cell surface and downstream pathways present on mRNA level. • Detected pathways indicate strong CHO survival and growth phenotypes. • Potential self-induction of surface receptors and respective ligands.

Purification and characterization of a fungal aspartic peptidase from Trichoderma reesei and its application for food and animal feed protein hydrolyses.

BACKGROUND: Various neutral and alkaline peptidases are commercially available for use in protein hydrolysis under neutral to alkaline conditions. However, the hydrolysis of proteins under acidic conditions by applying fungal aspartic peptidases (FAPs) has not been investigated in depth so far. The aim of this study, thus, was to purify a FAP from the commercial enzyme preparation, ROHALASE® BXL, determine its biochemical characteristics, and investigate its application for the hydrolysis of food and animal feed proteins under acidic conditions. RESULTS: A Trichoderma reesei derived FAP, with an apparent molecular mass of 45.8 kDa (sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SDS-PAGE) was purified 13.8-fold with a yield of 37% from ROHALASE® BXL. The FAP was identified as an aspartate protease (UniProt ID: G0R8T0) by inhibition and nano-LC-ESI-MS/MS studies. The FAP showed the highest activity at 50°C and pH 4.0. Monovalent cations, organic solvents, and reducing agents were tolerated well by the FAP. The FAP underwent an apparent competitive product inhibition by soy protein hydrolysate and whey protein hydrolysate with apparent Ki -values of 1.75 and 30.2 mg*mL-1 , respectively. The FAP showed promising results in food (soy protein isolate and whey protein isolate) and animal feed protein hydrolyses. For the latter, an increase in the soluble protein content of 109% was noted after 30 min. CONCLUSION: Our results demonstrate the applicability of fungal aspartic endopeptidases in the food and animal feed industry. Efficient protein hydrolysis of industrially relevant substrates such as acidic whey or animal feed proteins could be conducted by applying fungal aspartic peptidases. © 2022 Society of Chemical Industry.

The Phtheirospermum japonicum isopentenyltransferase PjIPT1a regulates host cytokinin responses in Arabidopsis.

The hemiparasitic plant Phtheirospermum japonicum (Phtheirospermum) is a nutritional specialist that supplements its nutrient requirements by parasitizing other plants through haustoria. During parasitism, the Phtheirospermum haustorium transfers hypertrophy-inducing cytokinins (CKs) to the infected host root. The CK biosynthesis genes required for haustorium-derived CKs and the induction of hypertrophy are still unknown. We searched for haustorium-expressed isopentenyltransferases (IPTs) that catalyze the first step of CK biosynthesis, confirmed the specific expression by in vivo imaging of a promoter-reporter, and further analyzed the subcellular localization, the enzymatic function and contribution to inducing hypertrophy by studying CRISPR-Cas9-induced Phtheirospermum mutants. PjIPT1a was expressed in intrusive cells of the haustorium close to the host vasculature. PjIPT1a and its closest homolog PjIPT1b located to the cytosol and showed IPT activity in vitro with differences in substrate specificity. Mutating PjIPT1a abolished parasite-induced CK responses in the host. A homolog of PjIPT1a also was identified in the related weed Striga hermonthica. With PjIPT1a, we identified the IPT enzyme that induces CK responses in Phtheirospermum japonicum-infected Arabidopsis roots. We propose that PjIPT1a exemplifies how parasitism-related functions evolve through gene duplications and neofunctionalization.

Unveiling the CHO surfaceome: Identification of cell surface proteins reveals cell aggregation-relevant mechanisms.

Chinese hamster ovary (CHO) suspension cells are the main production hosts for biopharmaceuticals. For the improvement of production processes, it is essential to understand the interaction between CHO cells and their microenvironment. While the cellular membrane is the crucial surface barrier between the inner and outer cell compartments, the subgroup of cell surface proteins (surfaceome) is of particular interest due to its potential to react to external factors and initiate cell communication and interaction pathways. Therefore, the CHO surfaceome was explored for the first time by enriching exposed N-glycosylated membrane proteins before tandem mass spectrometry (MS/MS) analyses, identifying a total of 449 surface proteins, including 34 proteins specific for production cells. Functional annotation and classification located most proteins to the cell surface belonging mainly to the protein classes of receptors, enzymes, and transporters. In addition, adhesion molecules as cadherins, integrins, Ig superfamily and extracellular matrix (ECM) proteins as collagens, laminins, thrombospondin, fibronectin, and tenascin were significantly enriched, which are involved in mechanisms for the formation of cell junctions, cell-cell and cell-ECM adhesion as focal adhesions. As cell adhesion and aggregation counteracts scalable production of biopharmaceuticals, experimental validation confirmed differential expression of integrin ß1 (ITGB1) and ß3, CD44, laminin, and fibronectin on the surface of aggregation-prone CHO production cells. The subsequent modulation of the central interaction protein ITGB1 by small interfering RNA knockdown substantially counteracted cell aggregation pointing toward novel engineering routes for aggregation reduction in biopharmaceutical production cells and exemplifying the potential of the surfaceome for specified engineering strategies.

Impact of Na+-Translocating NADH:Quinone Oxidoreductase on Iron Uptake and nqrM Expression in Vibrio cholerae.

The Na+ ion-translocating NADH:quinone oxidoreductase (NQR) from Vibrio cholerae is a membrane-bound respiratory enzyme which harbors flavins and Fe-S clusters as redox centers. The NQR is the main producer of the sodium motive force (SMF) and drives energy-dissipating processes such as flagellar rotation, substrate uptake, ATP synthesis, and cation-proton antiport. The NQR requires for its maturation, in addition to the six structural genes nqrABCDEF, a flavin attachment gene, apbE, and the nqrM gene, presumably encoding a Fe delivery protein. We here describe growth studies and quantitative real-time PCR for the V. cholerae O395N1 wild-type (wt) strain and its mutant Δnqr and ΔubiC strains, impaired in respiration. In a comparative proteome analysis, FeoB, the membrane subunit of the uptake system for Fe2+ (Feo), was increased in V. choleraeΔnqr In this study, the upregulation was confirmed on the mRNA level and resulted in improved growth rates of V. choleraeΔnqr with Fe2+ as an iron source. We studied the expression of feoB on other respiratory enzyme deletion mutants such as the ΔubiC mutant to determine whether iron transport is specific to the absence of NQR resulting from impaired respiration. We show that the nqr operon comprises, in addition to the structural nqrABCDEF genes, the downstream apbE and nqrM genes on the same operon and demonstrate induction of the nqr operon by iron in V. cholerae wt. In contrast, expression of the nqrM gene in V. choleraeΔnqr is repressed by iron. The lack of functional NQR has a strong impact on iron homeostasis in V. cholerae and demonstrates that central respiratory metabolism is interwoven with iron uptake and regulation.IMPORTANCE Investigating strategies of iron acquisition, storage, and delivery in Vibrio cholerae is a prerequisite to understand how this pathogen thrives in hostile, iron-limited environments such as the human host. In addition to highlighting the maturation of the respiratory complex NQR, this study points out the influence of NQR on iron metabolism, thereby making it a potential drug target for antibiotics.

Construction and description of a constitutive plipastatin mono-producing Bacillus subtilis.

BACKGROUND: Plipastatin is a potent Bacillus antimicrobial lipopeptide with the prospect to replace conventional antifungal chemicals for controlling plant pathogens. However, the application of this lipopeptide has so far been investigated in a few cases, principally because of the yield in low concentration and unknown regulation of biosynthesis pathways. B. subtilis synthesizes plipastatin by a non-ribosomal peptide synthetase encoded by the ppsABCDE operon. In this study, B. subtilis 3NA (a non-sporulation strain) was engineered to gain more insights about plipastatin mono-production. RESULTS: The 4-phosphopantetheinyl transferase Sfp posttranslationally converts non-ribosomal peptide synthetases from inactive apoforms into their active holoforms. In case of 3NA strain, sfp gene is inactive. Accordingly, the first step was an integration of a repaired sfp version in 3NA to construct strain BMV9. Subsequently, plipastatin production was doubled after integration of a fully expressed degQ version from B. subtilis DSM10T strain (strain BMV10), ensuring stimulation of DegU-P regulatory pathway that positively controls the ppsABSDE operon. Moreover, markerless substitution of the comparably weak native plipastatin promoter (Ppps) against the strong constitutive promoter Pveg led to approximately fivefold enhancement of plipastatin production in BMV11 compared to BMV9. Intriguingly, combination of both repaired degQ expression and promoter exchange (Ppps::Pveg) did not increase the plipastatin yield. Afterwards, deletion of surfactin (srfAA-AD) operon by the retaining the regulatory comS which is located within srfAB and is involved in natural competence development, resulted in the loss of plipastatin production in BMV9 and significantly decreased the plipastatin production of BMV11. We also observed that supplementation of ornithine as a precursor for plipastatin formation caused higher production of plipastatin in mono-producer strains, albeit with a modified pattern of plipastatin composition. CONCLUSIONS: This study provides evidence that degQ stimulates the native plipastatin production. Moreover, a full plipastatin production requires surfactin synthetase or some of its components. Furthermore, as another conclusion of this study, results point towards ornithine provision being an indispensable constituent for a plipastatin mono-producer B. subtilis strain. Therefore, targeting the ornithine metabolic flux might be a promising strategy to further investigate and enhance plipastatin production by B. subtilis plipastatin mono-producer strains.

Novel metagenome-derived ornithine lipids identified by functional screening for biosurfactants.

Biosurfactants are amphiphilic molecules that interact with the surfaces of liquids leading to many useful applications. Most biosurfactants have been identified from cultured microbial sources, leaving a largely untapped resource of uncultured bacteria with potentially novel biosurfactant structures. To access the uncultured bacteria, a metagenomic library was constructed in Escherichia coli from environmental DNA within an E. coli, Pseudomonas putida and Streptomyces lividans shuttle vector. Phenotypic screening of the library in E. coli and P. putida by the paraffin spray assay identified a P. putida clone with biosurfactant activity. Sequence analysis and transposon mutagenesis confirmed that an ornithine acyl-ACP N-acyltransferase was responsible for the activity. Although the fosmid was not active in E. coli, overexpression of the olsB gene could be achieved under the control of the inducible T7 promoter, resulting in lyso-ornithine lipid production and biosurfactant activity in the culture supernatants. Screening for activity in more than one host increases the range of sequences that can be identified through metagenomic, since olsB would not have been identified if only E. coli had been used as a host. The potential of lyso-ornithine lipids as a biosurfactant has not been fully explored. Here, we present several biosurfactant parameters of lyso-ornithine lipid to assess its suitability for industrial application.

Correction to: Novel metagenome-derived ornithine lipids identified by functional screening for biosurfactants.

The published online version contains mistake in the author list.

Metabolic Reprogramming of Vibrio cholerae Impaired in Respiratory NADH Oxidation Is Accompanied by Increased Copper Sensitivity.

The electrogenic, sodium ion-translocating NADH:quinone oxidoreductase (NQR) from Vibrio cholerae is frequent in pathogenic bacteria and a potential target for antibiotics. NQR couples the oxidation of NADH to the formation of a sodium motive force (SMF) and therefore drives important processes, such as flagellar rotation, substrate uptake, and energy-dissipating cation-proton antiport. We performed a quantitative proteome analysis of V. cholerae O395N1 compared to its variant lacking the NQR using minimal medium with glucose as the carbon source. We found 84 proteins (regulation factor of ≥2) to be changed in abundance. The loss of NQR resulted in a decrease in the abundance of enzymes of the oxidative branch of the tricarboxylic acid (TCA) cycle and an increase in abundance of virulence factors AcfC and TcpA. Most unexpected, the copper resistance proteins CopA, CopG, and CueR were decreased in the nqr deletion strain. As a consequence, the mutant exhibited diminished resistance to copper compared to the reference strain, as confirmed in growth studies using either glucose or mixed amino acids as carbon sources. We propose that the observed adaptations of the nqr deletion strain represent a coordinated response which counteracts a drop in transmembrane voltage that challenges V. cholerae in its different habitats.IMPORTANCE The importance of the central metabolism for bacterial virulence has raised interest in studying catabolic enzymes not present in the host, such as NQR, as putative targets for antibiotics. Vibrio cholerae lacking the NQR, which is studied here, is a model to estimate the impact of specific NQR inhibitors on the phenotype of a pathogen. Our comparative proteomic study provides a framework to evaluate the chances of success of compounds directed against NQR with respect to their bacteriostatic or bactericidal action.

Phytaspase-mediated precursor processing and maturation of the wound hormone systemin.

Peptide hormones are implicated in many important aspects of plant life and are usually synthesized as precursor proteins. In contrast to animals, data for plant peptide hormone maturation are scarce and the specificity of processing enzyme(s) is largely unknown. Here we tested a hypothesis that processing of prosystemin, a precursor of tomato (Solanum lycopersicum) wound hormone systemin, is performed by phytaspases, aspartate-specific proteases of the subtilase family. Following the purification of phytaspase from tomato leaves, two tomato phytaspase genes were identified, the cDNAs were cloned and the recombinant enzymes were obtained after transient expression in Nicotiana benthamiana. The newly identified tomato phytaspases hydrolyzed prosystemin at two aspartate residues flanking the systemin sequence. Site-directed mutagenesis of the phytaspase cleavage sites in prosystemin abrogated not only the phytaspase-mediated processing of the prohormone in vitro, but also the ability of prosystemin to trigger the systemic wound response in vivo. The data show that the prohormone prosystemin requires processing for signal biogenesis and biological activity. The identification of phytaspases as the proteases involved in prosystemin maturation provides insight into the mechanisms of wound signaling in tomato. Our data also suggest a novel role for cell death-related proteases in mediating defense signaling in plants.

A natural variant of arylsulfatase from Kluyveromyces lactis shows no formylglycine modification and has no enzyme activity.

Kluyveromyces lactis is a common fungal microorganism used for the production of enzyme preparations such as ß-galactosidases (native) or chymosin (recombinant). It is generally important that enzyme preparations have no unwanted side activities. In the case of ß-galactosidase preparations produced from K. lactis, an unwanted side activity could be the presence of arylsulfatase (EC 3.1.6.1). Due to the action of arylsulfatase, an unpleasant "cowshed-like" off-flavor would occur in the final product. The best choice to avoid this is to use a yeast strain without this activity. Interestingly, we found that certain natural K. lactis strains express arylsulfatases, which only differ in one amino acid at position 139. The result of this difference is that K. lactis DSM 70799 (expressing R139 variant) shows no arylsulfatase activity, unlike K. lactis GG799 (expressing S139 variant). After recombinant production of both variants in Escherichia coli, the R139 variant remains inactive, whereas the S139 variant showed full activity. Mass spectrometric analyses showed that the important posttranslational modification of C56 to formylglycine was not found in the R139 variant. By contrast, the C56 residue of the S139 variant was modified. We further investigated the packing and secondary structure of the arylsulfatase variants using optical spectroscopy, including fluorescence and circular dichroism. We found out that the inactive R139 variant exhibits a different structure regarding folding and packing compared to the active S139 variant. The importance of the amino acid residue 139 was documented further by the construction of 18 more variants, whereof only ten showed activity but always reduced compared to the native S139 variant.

Cell Death Control by Matrix Metalloproteinases.

In contrast to mammalian matrix metalloproteinases (MMPs) that play important roles in the remodeling of the extracellular matrix in animals, the proteases responsible for dynamic modifications of the plant cell wall are largely unknown. A possible involvement of MMPs was addressed by cloning and functional characterization of Sl2-MMP and Sl3-MMP from tomato (Solanum lycopersicum). The two tomato MMPs were found to resemble mammalian homologs with respect to gelatinolytic activity, substrate preference for hydrophobic amino acids on both sides of the scissile bond, and catalytic properties. In transgenic tomato seedlings silenced for Sl2/3-MMP expression, necrotic lesions were observed at the base of the hypocotyl. Cell death initiated in the epidermis and proceeded to include outer cortical cell layers. In later developmental stages, necrosis spread, covering the entire stem and extending into the leaves of MMP-silenced plants. The subtilisin-like protease P69B was identified as a substrate of Sl2- and Sl3-MMP. P69B was shown to colocalize with Sl-MMPs in the apoplast of the tomato hypocotyl, it exhibited increased stability in transgenic plants silenced for Sl-MMP activity, and it was cleaved and inactivated by Sl-MMPs in vitro. The induction of cell death in Sl2/3-MMP-silenced plants depended on P69B, indicating that Sl2- and Sl3-MMP act upstream of P69B in an extracellular proteolytic cascade that contributes to the regulation of cell death in tomato.

Expression in Pichia pastoris and characterization of two novel dirigent proteins for atropselective formation of gossypol.

We established an efficient fed-batch fermentation process for two novel dirigent proteins from cotton plants, GbDIR2 from Gossypium barbadense and GhDIR3 from G. hirsutum, using the engineered Pichia pastoris GlycoSwitch® SuperMan5 strain to prevent hyperglycosylation. The two (His)6-tagged proteins were purified by metal-chelate affinity chromatography and obtained in quantities of 12 and 15 mg L-1 of culture volume, respectively. Glycosylation sites were identified for the native and for the enzymatically deglycosylated proteins by mass spectrometry, confirming five to six of the seven predicted glycosylation sites in the NxS/T sequence context. The predominant glycan structure was Man5GlcNAc2 with, however, a significant contribution of Man4-10GlcNAc2. Both dirigent proteins (DIRs) mediated the formation of (+)-gossypol by atropselective coupling of hemigossypol radicals. Similar to previously characterized DIRs, GbDIR2 and GhDIR3 lacked oxidizing activity and depended on an oxidizing system (laccase/O2) for the generation of substrate radicals. In contrast to DIRs involved in the biosynthesis of lignans, glycosylation was not essential for function. Quantitative enzymatic deglycosylation yielded active GbDIR2 and GhDIR3 in excellent purity. The described fermentation process in combination with enzymatic deglycosylation will pave the way for mechanistic and structural studies and, eventually, the application of cotton DIRs in a biomimetic approach towards atropselective biaryl synthesis.

The Pseudomonas aeruginosa Isohexenyl Glutaconyl Coenzyme A Hydratase (AtuE) Is Upregulated in Citronellate-Grown Cells and Belongs to the Crotonase Family.

Pseudomonas aeruginosa is one of only a few Pseudomonas species that are able to use acyclic monoterpenoids, such as citronellol and citronellate, as carbon and energy sources. This is achieved by the acyclic terpene utilization pathway (Atu), which includes at least six enzymes (AtuA, AtuB, AtuCF, AtuD, AtuE, AtuG) and is coupled to a functional leucine-isovalerate utilization (Liu) pathway. Here, quantitative proteome analysis was performed to elucidate the terpene metabolism of P. aeruginosa. The proteomics survey identified 187 proteins, including AtuA to AtuG and LiuA to LiuE, which were increased in abundance in the presence of citronellate. In particular, two hydratases, AtuE and the PA4330 gene product, out of more than a dozen predicted in the P. aeruginosa proteome showed an increased abundance in the presence of citronellate. AtuE (isohexenyl-glutaconyl coenzyme A [CoA] hydratase; EC 4.2.1.57) most likely catalyzes the hydration of the unsaturated distal double bond in the isohexenyl-glutaconyl-CoA thioester to yield 3-hydroxy-3-isohexenyl-glutaryl-CoA. Determination of the crystal structure of AtuE at a 2.13-Å resolution revealed a fold similar to that found in the hydratase (crotonase) superfamily and provided insights into the nature of the active site. The AtuE active-site architecture showed a significantly broader cavity than other crotonase superfamily members, in agreement with the need to accommodate the branched isoprenoid unit of terpenes. Glu139 was identified to be a potential catalytic residue, while the backbone NH groups of Gly116 and Gly68 likely form an oxyanion hole. The present work deepens the understanding of terpene metabolism in Pseudomonas and may serve as a basis to develop new strategies for the biotechnological production of terpenoids.

Dirigent Proteins from Cotton (Gossypium sp.) for the Atropselective Synthesis of Gossypol.

Gossypol is a defense compound in cotton plants for protection against pests and pathogens. Gossypol biosynthesis involves the oxidative coupling of hemigossypol and results in two atropisomers owing to hindered rotation around the central binaphthyl bond. (+)-Gossypol predominates in vivo, thus suggesting stereochemically controlled biosynthesis. The aim was to identify the factors mediating (+)-gossypol formation in cotton and to investigate their potential for asymmetric biaryl synthesis. A dirigent protein from Gossypium hirsutum (GhDIR4) was found to confer atropselectivity to the coupling of hemigossypol in presence of laccase and O2 as an oxidizing agent. (+)-Gossypol was obtained in greater than 80% enantiomeric excess compared to racemic gossypol in the absence of GhDIR4. The identification of GhDIR4 highlights a broader role for DIRs in plant secondary metabolism and may eventually lead to the development of DIRs as tools for the synthesis of axially chiral binaphthyls.