Crystal structures of non-oxidative decarboxylases reveal a new mechanism of action with a catalytic dyad and structural twists.

Hydroxybenzoic acids, like gallic acid and protocatechuic acid, are highly abundant natural compounds. In biotechnology, they serve as critical precursors for various molecules in heterologous production pathways, but a major bottleneck is these acids' non-oxidative decarboxylation to hydroxybenzenes. Optimizing this step by pathway and enzyme engineering is tedious, partly because of the complicating cofactor dependencies of the commonly used prFMN-dependent decarboxylases. Here, we report the crystal structures (1.5-1.9 Å) of two homologous fungal decarboxylases, AGDC1 from Arxula adenivorans, and PPP2 from Madurella mycetomatis. Remarkably, both decarboxylases are cofactor independent and are superior to prFMN-dependent decarboxylases when heterologously expressed in Saccharomyces cerevisiae. The organization of their active site, together with mutational studies, suggests a novel decarboxylation mechanism that combines acid-base catalysis and transition state stabilization. Both enzymes are trimers, with a central potassium binding site. In each monomer, potassium introduces a local twist in a ß-sheet close to the active site, which primes the critical H86-D40 dyad for catalysis. A conserved pair of tryptophans, W35 and W61, acts like a clamp that destabilizes the substrate by twisting its carboxyl group relative to the phenol moiety. These findings reveal AGDC1 and PPP2 as founding members of a so far overlooked group of cofactor independent decarboxylases and suggest strategies to engineer their unique chemistry for a wide variety of biotechnological applications.

Artificial ER-Derived Vesicles as Synthetic Organelles for in Vivo Compartmentalization of Biochemical Pathways.

Compartmentalization in membrane-surrounded organelles has the potential to overcome obstacles associated with the engineering of metabolic pathways, such as unwanted side reactions, accumulation of toxic intermediates, drain of intermediates out of the cell, and long diffusion distances. Strategies utilizing natural organelles suffer from the presence of endogenous pathways. In our approach, we make use of endoplasmic reticulum-derived vesicles loaded with enzymes of a metabolic pathway ("metabolic vesicles"). They are generated by fusion of synthetic peptides containing the N-terminal proline-rich and self-assembling region of the maize storage protein gamma-Zein ("Zera") to the pathway enzymes. We have applied a strategy to integrate three enzymes of a cis,cis-muconic acid production pathway into those vesicles in yeast. Using fluorescence microscopy and cell fractionation techniques, we have proven the formation of metabolic vesicles and the incorporation of enzymes. Activities of the enzymes and functionality of the compartmentalized pathway were demonstrated in fermentation experiments.

An expanded enzyme toolbox for production of cis, cis-muconic acid and other shikimate pathway derivatives in Saccharomyces cerevisiae.

A wide range of commercially relevant aromatic chemicals can be synthesized via the shikimic acid pathway. Thus, this pathway has been the target of diverse metabolic engineering strategies. In the present work, an optimized yeast strain for production of the shikimic acid pathway intermediate 3-dehydroshikimate (3-DHS) was generated, which is a precursor for the production of the valuable compounds cis, cis-muconic acid (CCM) and gallic acid (GA). Production of CCM requires the overexpression of the heterologous enzymes 3-DHS dehydratase AroZ, protocatechuic acid (PCA) decarboxylase AroY and catechol dioxygenase CatA. The activity of AroY limits the yield of the pathway. This repertoire of enzymes was expanded by a novel fungal decarboxylase. Introducing this enzyme into the pathway in the optimized strain, a titer of 1244 mg L-1 CCM could be achieved, yielding 31 mg g-1 glucose. This represents the highest yield of this compound reported in Saccharomyces cerevisiae to date. To demonstrate the applicability of the optimized strain for production of other compounds from 3-DHS, we overexpressed AroZ together with a mutant of a para-hydroxybenzoic acid hydroxylase with improved substrate specificity for PCA, PobAY385F. Thereby, we could demonstrate the production of GA for the first time in S. cerevisiae.

Establishing a yeast-based screening system for discovery of human GLUT5 inhibitors and activators.

Human GLUT5 is a fructose-specific transporter in the glucose transporter family (GLUT, SLC2 gene family). Its substrate-specificity and tissue-specific expression make it a promising target for treatment of diabetes, metabolic syndrome and cancer, but few GLUT5 inhibitors are known. To identify and characterize potential GLUT5 ligands, we developed a whole-cell system based on a yeast strain deficient in fructose uptake, in which GLUT5 transport activity is associated with cell growth in fructose-based media or assayed by fructose uptake in whole cells. The former method is convenient for high-throughput screening of potential GLUT5 inhibitors and activators, while the latter enables detailed kinetic characterization of identified GLUT5 ligands. We show that functional expression of GLUT5 in yeast requires mutations at specific positions of the transporter sequence. The mutated proteins exhibit kinetic properties similar to the wild-type transporter and are inhibited by established GLUT5 inhibitors N-[4-(methylsulfonyl)-2-nitrophenyl]-1,3-benzodioxol-5-amine (MSNBA) and (-)-epicatechin-gallate (ECG). Thus, this system has the potential to greatly accelerate the discovery of compounds that modulate the fructose transport activity of GLUT5.

Pathway engineering for the production of heterologous aromatic chemicals and their derivatives in Saccharomyces cerevisiae: bioconversion from glucose.

Saccharomyces cerevisiae has been extensively engineered for optimising its performance as a microbial cell factory to produce valuable aromatic compounds and their derivatives as bulk and fine chemicals. The production of heterologous aromatic molecules in yeast is achieved via engineering of the aromatic amino acid biosynthetic pathway. This pathway is connected to two pathways of the central carbon metabolism, and is highly regulated at the gene and protein level. These characteristics impose several challenges for tailoring it, and various modifications need to be applied in order to redirect the carbon flux towards the production of the desired compounds. This minireview addresses the metabolic engineering approaches targeting the central carbon metabolism, the shikimate pathway and the tyrosine and phenylalanine biosynthetic pathway of S. cerevisiae for biosynthesis of aromatic chemicals and their derivatives from glucose.

Requirement of a Functional Flavin Mononucleotide Prenyltransferase for the Activity of a Bacterial Decarboxylase in a Heterologous Muconic Acid Pathway in Saccharomyces cerevisiae.

Biotechnological production of cis,cis-muconic acid from renewable feedstocks is an environmentally sustainable alternative to conventional, petroleum-based methods. Even though a heterologous production pathway for cis,cis-muconic acid has already been established in the host organism Saccharomyces cerevisiae, the generation of industrially relevant amounts of cis,cis-muconic acid is hampered by the low activity of the bacterial protocatechuic acid (PCA) decarboxylase AroY isomeric subunit Ciso (AroY-Ciso), leading to secretion of large amounts of the intermediate PCA into the medium. In the present study, we show that the activity of AroY-Ciso in S. cerevisiae strongly depends on the strain background. We could demonstrate that the strain dependency is caused by the presence or absence of an intact genomic copy of PAD1, which encodes a mitochondrial enzyme responsible for the biosynthesis of a prenylated form of the cofactor flavin mononucleotide (prFMN). The inactivity of AroY-Ciso in strain CEN.PK2-1 could be overcome by plasmid-borne expression of Pad1 or its bacterial homologue AroY subunit B (AroY-B). Our data reveal that the two enzymes perform the same function in decarboxylation of PCA by AroY-Ciso, although coexpression of Pad1 led to higher decarboxylase activity. Conversely, AroY-B can replace Pad1 in its function in decarboxylation of phenylacrylic acids by ferulic acid decarboxylase Fdc1. Targeting of the majority of AroY-B to mitochondria by fusion to a heterologous mitochondrial targeting signal did not improve decarboxylase activity of AroY-Ciso, suggesting that mitochondrial localization has no major impact on cofactor biosynthesis.IMPORTANCE In Saccharomyces cerevisiae, the decarboxylation of protocatechuic acid (PCA) to catechol is the bottleneck reaction in the heterologous biosynthetic pathway for production of cis,cis-muconic acid, a valuable precursor for the production of bulk chemicals. In our work, we demonstrate the importance of the strain background for the activity of a bacterial PCA decarboxylase in S. cerevisiae Inactivity of the decarboxylase is due to a nonsense mutation in a gene encoding a mitochondrial enzyme involved in the biosynthesis of a cofactor required for decarboxylase function. Our study reveals functional interchangeability of Pad1 and a bacterial homologue, irrespective of their intracellular localization. Our results open up new possibilities to improve muconic acid production by engineering cofactor supply. Furthermore, the results have important implications for the choice of the production strain.

Secretome analysis of Anabaena sp. PCC 7120 and the involvement of the TolC-homologue HgdD in protein secretion.

Secretion of proteins is a central strategy of bacteria to influence and respond to their environment. Until now, there has been very few discoveries regarding the cyanobacterial secrotome or the secretion machineries involved. For a mutant of the outer membrane channel TolC-homologue HgdD of Anabaena sp. PCC 7120, a filamentous and heterocyst-forming cyanobacterium, an altered secretome profile was reported. To define the role of HgdD in protein secretion, we have developed a method to isolate extracellular proteins of Anabaena sp. PCC 7120 wild type and an hgdD loss-of-function mutant. We identified 51 proteins of which the majority is predicted to have an extracellular secretion signal, while few seem to be localized in the periplasmic space. Eight proteins were exclusively identified in the secretome of wild-type cells, which coincides with the distribution of type I secretion signal. We selected three candidates and generated hemagglutinin-tagged fusion proteins which could be exclusively detected in the extracellular protein fraction. However, these proteins are not secreted in the hgdD-mutant background, where they are rapidly degraded. This confirms a direct function of HgdD in protein secretion and points to the existence of a quality control mechanism at least for proteins secreted in an HgdD-dependent pathway.

In vivo function of Tic22, a protein import component of the intermembrane space of chloroplasts.

Preprotein import into chloroplasts depends on macromolecular machineries in the outer and inner chloroplast envelope membrane (TOC and TIC). It was suggested that both machineries are interconnected by components of the intermembrane space (IMS). That is, amongst others, Tic22, of which two closely related isoforms exist in Arabidopsis thaliana, namely atTic22-III and atTic22-IV. We investigated the function of Tic22 in vivo by analyzing T-DNA insertion lines of the corresponding genes. While the T-DNA insertion in the individual genes caused only slight defects, a double mutant of both isoforms showed retarded growth, a pale phenotype under high-light conditions, a reduced import rate, and a reduction in the photosynthetic performance of the plants. The latter is supported by changes in the metabolite content of mutant plants when compared to wild-type. Thus, our results support the notion that Tic22 is directly involved in chloroplast preprotein import and might point to a particular importance of Tic22 in chloroplast biogenesis at times of high import rates.

Toc33 and Toc64-III cooperate in precursor protein import into the chloroplasts of Arabidopsis thaliana.

The import of cytosolically synthesized precursor proteins into chloroplasts by the translocon at the outer envelope membrane of chloroplasts (TOC) is crucial for organelle function. The recognition of precursor proteins at the chloroplast surface precedes translocation and involves the membrane-inserted receptor subunits Toc34 and Toc159. A third receptor, Toc64, was discussed to recognize cytosolic complexes guiding precursor proteins to the membrane surface, but this function remains debated. We analysed Arabidopsis thaliana plants carrying a T-DNA insertion in the gene encoding the Toc64 homolog Toc64-III. We observed a light intensity-dependent growth phenotype, which is distinct from the phenotype of ppi1, the previously described mutant of the TOC34 homolog TOC33. Furthermore, chloroplast import of the model precursor proteins pOE33 and pSSU into chloroplasts is reduced in protoplasts isolated from plants with impaired Toc64-III function. This suggests that Toc64-III modulates the translocation efficiency in vivo. A ppi1 and toc64-III double mutant shows a significant increase in the transcript levels of HSP90 and TOC75-III, the latter coding for the pore-forming TOC component. Remarkably, the protein level of Toc75-III is significantly reduced, suggesting that Toc64-III and Toc33 cooperate in the insertion or stabilization of Toc75-III. Accordingly, the results presented support Toc64 as an import-relevant component of the TOC complex.

Structure and conservation of the periplasmic targeting factor Tic22 protein from plants and cyanobacteria.

Mitochondria and chloroplasts are of endosymbiotic origin. Their integration into cells entailed the development of protein translocons, partially by recycling bacterial proteins. We demonstrate the evolutionary conservation of the translocon component Tic22 between cyanobacteria and chloroplasts. Tic22 in Anabaena sp. PCC 7120 is essential. The protein is localized in the thylakoids and in the periplasm and can be functionally replaced by a plant orthologue. Tic22 physically interacts with the outer envelope biogenesis factor Omp85 in vitro and in vivo, the latter exemplified by immunoprecipitation after chemical cross-linking. The physical interaction together with the phenotype of a tic22 mutant comparable with the one of the omp85 mutant indicates a concerted function of both proteins. The three-dimensional structure allows the definition of conserved hydrophobic pockets comparable with those of ClpS or BamB. The results presented suggest a function of Tic22 in outer membrane biogenesis.

The localization of Tic20 proteins in Arabidopsis thaliana is not restricted to the inner envelope membrane of chloroplasts.

Tic20 is a central, membrane-embedded component of the precursor protein translocon of the inner envelope of chloroplasts (TIC). In Arabidopsis thaliana, four different isoforms of Tic20 exist. They are annotated as atTic20-I, -II, -IV and -V and form two distinct phylogenetic subfamilies in embryophyta. Consistent with atTic20-I being the only essential isoform for chloroplast development, we show that the protein is exclusively targeted to the chloroplasts inner envelope. The same result is observed for atTic20-II. In contrast, atTic20-V is localized in thylakoids and atTic20-IV dually localizes to chloroplasts and mitochondria. These results together with the previously established expression profiles explain the recently described phenotypes of Tic20 knockout plants and point towards a functional diversification of these proteins within the family. For all Tic20 proteins a 4-helix topology is proposed irrespective of the targeted membrane, which in part could be confirmed in vivo by application of a self-assembling GFP-based topology approach. By the same approach we show that the inner envelope localized Tic20 proteins expose their C-termini to the chloroplast stroma. This localization would be consistent with the positive inside rule considering a stromal translocation intermediate as discussed.

Studying chloroplast protein interactions in vitro: an overview of the available methods.

The analysis of protein-protein interactions is essential for the understanding of the molecular events in enzymatic pathways, signaling cascades, or transport processes in the chloroplast. A large variety of methods are available, which range from qualitative assays allowing for screening for new interaction partners, and semiquantitative assays allowing for a rough description of the interaction between two partners, to quantitative assays that permit detailed determination of kinetic and thermodynamic parameters. We summarize the available technologies, describe their range of applications and pitfalls, and give some examples from chloroplast research. The described techniques are generic and thereby important and useful to study the interaction network of proteins in Arabidopsis thaliana. In addition, we refer the reader to detailed protocols published elsewhere for each method.

Functional dissection of the cytosolic chaperone network in tomato mesophyll protoplasts.

The heat stress response is universal to all organisms. Upon elevated temperatures, heat stress transcription factors (Hsfs) are activated to up-regulate the expression of molecular chaperones to protect cells against heat damages. In higher plants, the phenomenon is unusually complex both at the level of Hsfs and heat stress proteins (Hsps). Over-expression of both Hsfs and Hsps and the use of RNA interference for gene knock-down in a transient system in tomato protoplasts allowed us to dissect the in vivo chaperone functions of essential components of thermotolerance, such as the cytoplasmic sHsp, Hsp70 and Hsp100 chaperone families, and the regulation of their expression. The results point to specific functions of the different components in protection from protein denaturation and in refolding of denatured proteins.

A novel serine/proline-rich domain in combination with a transmembrane domain is required for the insertion of AtTic40 into the inner envelope membrane of chloroplasts.

AtTic40 is part of the chloroplastic protein import apparatus that is anchored in the inner envelope membrane by a single N-terminal transmembrane domain, and has a topology in which the bulk of the C-terminal domain is oriented toward the stroma. The targeting of AtTic40 to the inner envelope membrane involves two steps. Using an in vitro import assay, we showed that the sorting of AtTic40 requires a bipartite transit peptide, which was first cleaved by the stromal processing peptidase (SPP), thus generating a soluble AtTic40 stromal intermediate (iAtTic40). iAtTic40 was further processed by a second unknown peptidase, which generates its mature form (mAtTic40). Using deletion mutants, we identified a sequence motif N-terminal of the transmembrane domain that was essential for reinsertion of iAtTic40 into the inner envelope membrane. We have designated this region a serine/proline-rich (S/P-rich) domain and present a model describing its role in the targeting of AtTic40 to the inner envelope membrane.

Immunomodulation of function of small heat shock proteins prevents their assembly into heat stress granules and results in cell death at sublethal temperatures.

The conformational dynamism and aggregate state of small heat shock proteins (sHSPs) may be crucial for their functions in thermoprotection of plant cells from the detrimental effects of heat stress. Ectopic expression of single chain fragment variable (scFv) antibodies against cytosolic sHSPs was used as new tool to generate sHSP loss-of-function mutants by antibody-mediated prevention of the sHSP assembly in vivo. Anti-sHSP scFv antibodies transiently expressed in heat-stressed tobacco protoplasts were not only able to recognize the endogenous sHSPs but also prevented their assembly into heat stress granula (HSGs). Constitutive expression of the same scFv antibodies in transgenic plants did not alter their phenotype at normal growth temperatures, but their leaves turned yellow and died after prolonged stress at sublethal temperatures. Structural analysis revealed a regular cytosolic distribution of stress-induced sHSPs in mesophyll cells of stress-treated transgenic plants, whereas extensive formation of HSGs was observed in control cells. After prolonged stress at sublethal temperatures, mesophyll cells of transgenic plants suffered destruction of all cellular membranes and finally underwent cell death. In contrast, mesophyll cells of the stressed controls showed HSG disintegration accompanied by appearance of polysomes, dictyosomes and rough endoplasmic reticulum indicating normalization of cell functions. Apparently, the ability of sHSPs to assemble into HSGs as well as the HSG disintegration is a prerequisite for survival of plant cells under continuous stress conditions at sublethal temperatures.

Heat stress response in plants: a complex game with chaperones and more than twenty heat stress transcription factors.

Compared to the overall multiplicity of more than 20 plant Hsfs, detailed analyses are mainly restricted to tomato and Arabidopsis and to three important representatives of the family (Hsfs A1, A2 and B1). The three Hsfs represent examples of striking functional diversification specialized for the three phases of the heat stress (hs) response (triggering, maintenance and recovery). This is best illustrated for the tomato Hsf system: (i) HsfA1a is the master regulator responsible for hs-induced gene expression including synthesis of HsfA2 and HsfB1. It is indispensible for the development of thermotolerance. (ii) Although functionally equivalent to HsfA1a, HsfA2 is exclusively found after hs induction and represents the dominant Hsf, the "working horse" of the hs response in plants subjected to repeated cycles of hs and recovery in a hot summer period. Tomato HsfA2 is tightly integrated into a network of interacting proteins (HsfA1a, Hsp17-CII, Hsp17-CI) influencing its activity and intracellular distribution. (iii) Because of structural peculiarities, HsfB1 acts as coregulator enhancing the activity of HsfA1a and/or HsfA2. But in addition, it cooperates with yet to be identified other transcription factors in maintaining and/or restoring housekeeping gene expression.

Role of Hsp17.4-CII as coregulator and cytoplasmic retention factor of tomato heat stress transcription factor HsfA2.

HsfA2 is a heat stress (hs)-induced Hsf in peruvian tomato (Lycopersicon peruvianum) and the cultivated form Lycopersicon esculentum. Due to the high activator potential and the continued accumulation during repeated cycles of heat stress and recovery, HsfA2 becomes a dominant Hsf in thermotolerant cells. The formation of heterooligomeric complexes with HsfA1 leads to nuclear retention and enhanced transcriptional activity of HsfA2. This effect seems to represent one part of potential molecular mechanisms involved in its activity control. As shown in this paper, the activity of HsfA2 is also controlled by a network of nucleocytoplasmic small Hsps influencing its solubility, intracellular localization and activator function. By yeast two-hybrid interaction and transient coexpression studies in tobacco (Nicotiana plumbaginifolia) mesophyll protoplasts, we found that tomato (Lycopersicon esculentum) Hsp17.4-CII acts as corepressor of HsfA2. Given appropriate conditions, both proteins together formed large cytosolic aggregates which could be solubilized in presence of class CI sHsps. However, independent of the formation of aggregates or of the nucleocytoplasmic distribution of HsfA2, its transcriptional activity was specifically repressed by interaction of Hsp17.4-CII with the C-terminal activator domain. Although not identical in all aspects, the situation with the highly expressed, heat stress-inducible Arabidopsis HsfA2 was found to be principally similar. In corresponding reporter assays its activity was repressed in presence of AtHsp17.7-CII but not of AtHsp17.6-CII or LpHsp17.4-CII.

Tomato heat stress protein Hsp16.1-CIII represents a member of a new class of nucleocytoplasmic small heat stress proteins in plants.

We describe a new class of plant small heat stress proteins (sHsps) with dominant nuclear localization (Hsp17-CIII). The corresponding proteins in tomato, Arabidopsis, and rice are encoded by unique genes containing a short intron in the beta4-encoding region of the alpha-crystallin domain (ACD). The strong nuclear localization results from a cluster of basic amino acid residues in the loop between beta5 and beta6 of the ACD. Using yeast 2-hybrid tests, analyses of native complexes of the sHsps, and immunofluorescence data, we demonstrate that, in contrast to earlier observations (Kirschner et al 2000), proteins of the sHsp classes CI, CII, and CIII interact with each other, thereby influencing oligomerization state and intracellular localization.

In the complex family of heat stress transcription factors, HsfA1 has a unique role as master regulator of thermotolerance in tomato.

We generated transgenic tomato plants with altered expression of heat stress transcription factor HsfA1. Plants with 10-fold overexpression of HsfA1 (OE plants) were characterized by a single HsfA1 transgene cassette, whereas plants harboring a tandem inverted repeat of the cassette showed cosuppression (CS plants) by posttranscriptional silencing of the HsfA1 gene connected with formation of small interfering RNAs. Under normal growth conditions, major developmental parameters were similar for wild-type (WT), OE, and CS plants. However, CS plants and fruits were extremely sensitive to elevated temperatures, because heat stress-induced synthesis of chaperones and Hsfs was strongly reduced or lacking. Despite the complexity of the plant Hsf family with at least 17 members in tomato, HsfA1 has a unique function as master regulator for induced thermotolerance. Using transient reporter assays with mesophyll protoplasts from WT tomato, we demonstrated that plasmid-encoded HsfA1 and HsfA2 were well expressed. However, in CS protoplasts the cosuppression phenomenon was faithfully reproduced. Only transformation with HsfA2 expression plasmid led to normal expression of the transcription factor and reporter gene activation, whereas even high amounts of HsfA1 expression plasmids were silenced. Thermotolerance in CS protoplasts was restored by plasmid-borne HsfA2, resulting in expression of chaperones, thermoprotection of firefly luciferase, and assembly of heat stress granules.