Determining the Stoichiometry of Small Protein Oligomers Using Steady-State Fluorescence Anisotropy.

A large fraction of soluble and membrane-bound proteins exists as non-covalent dimers, trimers, and higher-order oligomers. The experimental determination of the oligomeric state or stoichiometry of proteins remains a nontrivial challenge. In one approach, the protein of interest is genetically fused to green fluorescent protein (GFP). If a fusion protein assembles into a non-covalent oligomeric complex, exciting their GFP moiety with polarized fluorescent light elicits homotypic Förster resonance energy transfer (homo-FRET), in which the emitted radiation is partially depolarized. Fluorescence depolarization is associated with a decrease in fluorescence anisotropy that can be exploited to calculate the oligomeric state. In a classical approach, several parameters obtained through time-resolved and steady-state anisotropy measurements are required for determining the stoichiometry of the oligomers. Here, we examined novel approaches in which time-resolved measurements of reference proteins provide the parameters that can be used to interpret the less expensive steady-state anisotropy data of candidates. In one approach, we find that using average homo-FRET rates (kFRET), average fluorescence lifetimes (τ), and average anisotropies of those fluorophores that are indirectly excited by homo-FRET (rET) do not compromise the accuracy of calculated stoichiometries. In the other approach, fractional photobleaching of reference oligomers provides a novel parameter a whose dependence on stoichiometry allows one to quantitatively interpret the increase of fluorescence anisotropy seen after photobleaching the candidates. These methods can at least reliably distinguish monomers from dimers and trimers.

Role of GxxxG Motifs in Transmembrane Domain Interactions.

Transmembrane (TM) helices of integral membrane proteins can facilitate strong and specific noncovalent protein-protein interactions. Mutagenesis and structural analyses have revealed numerous examples in which the interaction between TM helices of single-pass membrane proteins is dependent on a GxxxG or (small)xxx(small) motif. It is therefore tempting to use the presence of these simple motifs as an indicator of TM helix interactions. In this Current Topic review, we point out that these motifs are quite common, with more than 50% of single-pass TM domains containing a (small)xxx(small) motif. However, the actual interaction strength of motif-containing helices depends strongly on sequence context and membrane properties. In addition, recent studies have revealed several GxxxG-containing TM domains that interact via alternative interfaces involving hydrophobic, polar, aromatic, or even ionizable residues that do not form recognizable motifs. In multipass membrane proteins, GxxxG motifs can be important for protein folding, and not just oligomerization. Our current knowledge thus suggests that the presence of a GxxxG motif alone is a weak predictor of protein dimerization in the membrane.

Maximized Autotransporter-Mediated Expression (MATE) for Surface Display and Secretion of Recombinant Proteins in Escherichia coli.

A new optimized system for the surface display and secretion of recombinant proteins is described, termed MATE (maximized autotransporter-mediated expression). It is based on an artificial gene consisting of the coding region for the signal peptide of CtxB, a multiple cloning site for passenger gene insertion, flanked by coding sequences for linear epitopes for monoclonal antibodies and OmpT, and factor Xa protease cleavage sites followed by a codon-optimized DNA sequence of the linker and the ß-barrel of the type V autotransporter EhaA from Escherichia coli under control of an IPTG-inducible T5 promoter. The MATE system enabled the continuous secretion of recombinant passenger mCherry via OmpT-mediated cleavage, using native OmpT protease activity in E. coli when grown at 37 °C. It is the first example to show that native OmpT activity is sufficient to facilitate the secretion of a correctly folded target protein in preparative amounts obtaining 240 µg of purified mCherry from 800 mL of crude culture supernatant. Because the release of mCherry was achieved by a simple transfer of the encoding plasmid from an OmpT-negative to an OmpT-positive strain, it bears the option to use surface display for screening purposes and secretion for production of the selected variant. A single plasmid could therefore be used for continuous secretion in OmpT-positive strains or surface display in OmpT-negative strains. In conclusion, the MATE system appears to be a versatile tool for the surface display and for the secretion of target proteins in E. coli.

Escherichia coli kduD encodes an oxidoreductase that converts both sugar and steroid substrates.

A previously unidentified oxidoreductase from Escherichia coli catalyzes the regioselective reduction of eukaryotic steroid hormone 11-deoxycorticosterone (11-DOC) to the valuable bioactive product 4-pregnen-20,21-diol-3-one. In nature, a reduction of C-20 carbonyl of C21 steroids is catalyzed by diverse NAD(P)H-dependent oxidoreductases. Enzymes that possess 20-ketosteroid reductase activity, however, have never before been described in E. coli. Our present study aimed to identify and characterize the E. coli enzyme which possesses 20-ketosteroid reductase activity against eukaryotic steroid hormone 11-DOC. We partially purified the enzyme from E. coli DH5α using protein chromatography techniques. Mass spectrometry revealed the presence of three NADH-specific oxidoreductases in the sample. The genes encoding these oxidoreductases were cloned and overexpressed in E. coli UT5600 (DE3). Only the overexpression of 2-dehydro-3-deoxy-D-gluconate 5-dehydrogenase (KduD) encoded by kduD gene enabled the whole-cell biotransformation of 11-DOC. A 6xHis-tagged version of KduD was purified to homogeneity and found to reduce several eukaryotic steroid hormones and catalyze the conversion of novel sugar substrates. KduD from E. coli is therefore a promiscuous enzyme that has a predicted role in sugar conversion in vivo but can be used for the production of valuable bioactive 20-hydroxysteroids.

Synthesis and biological evaluation of flexible and conformationally constrained LpxC inhibitors.

Inhibitors of the UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC) represent promising candidates for the development of antibiotics possessing a so far unexploited mechanism of action. In a chiral pool synthesis, starting from the D-mannose derived mannonolactone 4, conformationally constrained C-glycosidic as well as open chained hydroxamic acids with a defined stereochemistry were prepared. Diversity was introduced by performing C­C coupling reactions like the Sonogashira and Suzuki cross-coupling reactions. The biological evaluation of the synthesized compounds revealed that in the case of the C-glycosides a long, linear and rigid hydrophobic side chain is required for antibiotic activity against E. coli. The open chain derivatives show higher biological activity than the conformationally constrained C-glycosides. The morpholinomethyl substituted open chain derivative 43, being the most potent compound presented in this paper, inhibits LpxC with a Ki value of 0.35 µM and represents a promising lead structure.

Experimental determination and data-driven prediction of homotypic transmembrane domain interfaces.

Interactions between their transmembrane domains (TMDs) frequently support the assembly of single-pass membrane proteins to non-covalent complexes. Yet, the TMD-TMD interactome remains largely uncharted. With a view to predicting homotypic TMD-TMD interfaces from primary structure, we performed a systematic analysis of their physical and evolutionary properties. To this end, we generated a dataset of 50 self-interacting TMDs. This dataset contains interfaces of nine TMDs from bitopic human proteins (Ire1, Armcx6, Tie1, ATP1B1, PTPRO, PTPRU, PTPRG, DDR1, and Siglec7) that were experimentally identified here and combined with literature data. We show that interfacial residues of these homotypic TMD-TMD interfaces tend to be more conserved, coevolved and polar than non-interfacial residues. Further, we suggest for the first time that interface positions are deficient in ß-branched residues, and likely to be located deep in the hydrophobic core of the membrane. Overrepresentation of the GxxxG motif at interfaces is strong, but that of (small)xxx(small) motifs is weak. The multiplicity of these features and the individual character of TMD-TMD interfaces, as uncovered here, prompted us to train a machine learning algorithm. The resulting prediction method, THOIPA (www.thoipa.org), excels in the prediction of key interface residues from evolutionary sequence data.

Author Correction: Identifying ionic interactions within a membrane using BLaTM, a genetic tool to measure homo- and heterotypic transmembrane helix-helix interactions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The enzymatic basis for pesticide bioremediation.

Enzymes are central to the biology of many pesticides, influencing their modes of action, environmental fates and mechanisms of target species resistance. Since the introduction of synthetic xenobiotic pesticides, enzymes responsible for pesticide turnover have evolved rapidly, in both the target organisms and incidentally exposed biota. Such enzymes are a source of significant biotechnological potential and form the basis of several bioremediation strategies intended to reduce the environmental impacts of pesticide residues. This review describes examples of enzymes possessing the major activities employed in the bioremediation of pesticide residues, and some of the strategies by which they are employed. In addition, several examples of specific achievements in enzyme engineering are considered, highlighting the growing trend in tailoring enzymatic activity to a specific biotechnologically relevant function.

Surface modification of poly(hydroxybutyrate) films to control cell-matrix adhesion.

Tailoring surface properties of degradable polymer scaffolds is key to progress in various tissue engineering strategies. Poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) thin films were modified by low pressure ammonia plasma, low pressure water vapour plasma, or immersion in a sodium hydroxide solution to elaborate means to control the cell-matrix adhesion of human umbilical cord vein endothelial cells grown on these materials. Fibronectin (FN) heteroexchange and cell adhesion were correlated to the physicochemical characteristics of the modified polymer surfaces which were investigated by X-ray photoelectron spectroscopy (XPS), scanning force microscopy (SFM), electrokinetic measurements, and contact angle measurements. All treatments increased the hydrophilicity of the polymer samples, which could be accounted to newly created amine or carboxyl functionalities for ammonia plasma or water vapour plasma treatments, respectively, and ester hydrolysis for treatments with alkaline aqueous solutions. Main features of cell adhesion and FN reorganisation-evaluated after 1h and after 5 days-could be attributed to the anchorage strength of pre-coated FN layers at the polymer surface, which was, in turn found to be triggered by the type of modification applied. In line with earlier studies referring to different materials cell adhesion and matrix reorganisation were shown to be sensitively controlled through the physicochemical profile of poly(hydroxybutyrate) surfaces.

Identifying ionic interactions within a membrane using BLaTM, a genetic tool to measure homo- and heterotypic transmembrane helix-helix interactions.

The assembly of integral membrane protein complexes is frequently supported by transmembrane domain (TMD) interactions. Here, we present the BLaTM assay that measures homotypic as well as heterotypic TMD-TMD interactions in a bacterial membrane. The system is based on complementation of ß-lactamase fragments genetically fused to interacting TMDs, which confers ampicillin resistance to expressing cells. We validated BLaTM by showing that the assay faithfully reports known sequence-specific interactions of both types. In a practical application, we used BLaTM to screen a focussed combinatorial library for heterotypic interactions driven by electrostatic forces. The results reveal novel patterns of ionizable amino acids within the isolated TMD pairs. Those patterns indicate that formation of heterotypic TMD pairs is most efficiently supported by closely spaced ionizable residues of opposite charge. In addition, TMD heteromerization can apparently be driven by hydrogen bonding between basic or between acidic residues.

Autotransporter mediated esterase display on Zymomonas mobilis and Zymobacter palmae.

Autodisplay, i.e. surface expression of recombinant proteins by virtue of the autotransporter secretion pathway, has been used predominantly with Escherichia coli as host organism, which often limits the applicability of this technique to laboratory purposes and scales. The aim of this study was to investigate if the fermentative bacteria Zymomonas mobilis and Zymobacter palmae, representing attractive candidates for industrial applications, can serve as host organisms for autodisplay. We therefore used the carboxylesterase EstA from Burkholderia gladioli as an autotransporter passenger to display it on the surfaces of Z. palmae and Z. mobilis. Expression and outer membrane localization of the EstA-autotransporter fusion protein were verified by SDS-PAGE, and surface display of the enzyme was demonstrated by ELISA and flow cytometer analysis. Whole-cell activity assays revealed that EstA retained its activity on the cell surface. Recombinant Z. palmae cells exhibited significant higher esterase activity (294mU/mL/OD 1) in comparison to Z. mobilis (88mU/mL/OD 1) and the control E. coli (113mU/mL/OD 1). This appears even more noteworthy, as about 30% of EstA was released from the cell surface of Z. palmae. Nevertheless, our results indicate that both species are suitable autodisplay hosts, in particular Z. palmae for displaying esterase, opening up new horizons for biocatalytic applications.

Heterologous expression and biochemical characterisation of fourteen esterases from Helicoverpa armigera.

Esterases have recurrently been implicated in insecticide resistance in Helicoverpa armigera but little is known about the underlying molecular mechanisms. We used a baculovirus system to express 14 of 30 full-length esterase genes so far identified from midgut cDNA libraries of this species. All 14 produced esterase isozymes after native PAGE and the isozymes for seven of them migrated to two regions of the gel previously associated with both organophosphate and pyrethroid resistance in various strains. Thirteen of the enzymes obtained in sufficient yield for further analysis all showed tight binding to organophosphates and low but measurable organophosphate hydrolase activity. However there was no clear difference in activity between the isozymes from regions associated with resistance and those from elsewhere in the zymogram, or between eight of the isozymes from a phylogenetic clade previously associated with resistance in proteomic and quantitative rtPCR experiments and five others not so associated. By contrast, the enzymes differed markedly in their activities against nine pyrethroid isomers and the enzymes with highest activity for the most insecticidal isomers were from regions of the gel and, in some cases, the phylogeny that had previously been associated with pyrethroid resistance. Phospholipase treatment confirmed predictions from sequence analysis that three of the isozymes were GPI anchored. This unusual feature among carboxylesterases has previously been suggested to underpin an association that some authors have noted between esterases and resistance to the Cry1Ac toxin from Bacillus thuringiensis. However these three isozymes did not migrate to the zymogram region previously associated with Cry1Ac resistance.

Organophosphate and pyrethroid hydrolase activities of mutant Esterases from the cotton bollworm Helicoverpa armigera.

Two mutations have been found in five closely related insect esterases (from four higher Diptera and a hymenopteran) which each confer organophosphate (OP) hydrolase activity on the enzyme and OP resistance on the insect. One mutation converts a Glycine to an Aspartate, and the other converts a Tryptophan to a Leucine in the enzymes' active site. One of the dipteran enzymes with the Leucine mutation also shows enhanced activity against pyrethroids. Introduction of the two mutations in vitro into eight esterases from six other widely separated insect groups has also been reported to increase substantially the OP hydrolase activity of most of them. These data suggest that the two mutations could contribute to OP, and possibly pyrethroid, resistance in a variety of insects. We therefore introduced them in vitro into eight Helicoverpa armigera esterases from a clade that has already been implicated in OP and pyrethroid resistance. We found that they do not generally enhance either OP or pyrethroid hydrolysis in these esterases but the Aspartate mutation did increase OP hydrolysis in one enzyme by about 14 fold and the Leucine mutation caused a 4-6 fold increase in activity (more in one case) of another three against some of the most insecticidal isomers of fenvalerate and cypermethrin. The Aspartate enzyme and one of the Leucine enzymes occur in regions of the H. armigera esterase isozyme profile that have been previously implicated in OP and pyrethroid resistance, respectively.

Autodisplay of enzymes--molecular basis and perspectives.

To display an enzyme on the surface of a living cell is an important step forward towards a broader use of biocatalysts. Enzymes immobilized on surfaces appeared to be more stable compared to free molecules. It is possible by standard techniques to let the bacterial cell (e.g. Escherichia coli) decorate its surface with the enzyme and produce it on high amounts with a minimum of costs and equipment. Moreover, these cells can be recovered and reused in several subsequent process cycles. Among other systems, autodisplay has some extra features that could overcome limitations in the industrial applications of enzymes. One major advantage of autodisplay is the motility of the anchoring domain. Enzyme subunits exposed at the cell surface having affinity to each other will spontaneously form dimers or multimers. Using autodisplay enzymes with prosthetic groups can be displayed, expanding the application of surface display to the industrial important P450 enzymes. Finally, up to 105-106 enzyme molecules can be displayed on a single cell. In the present review, we summarize recent achievements in the autodisplay of enzymes with particular attention to industrial needs and process development. Applications that will provide sustainable solutions towards a bio-based industry are discussed.

Autodisplay of functional CYP106A2 in Escherichia coli.

Cytochrome P450 enzymes catalyse a wide variety of reactions, including the hydroxylation and epoxidation of CC bonds, and dealkylation reactions. There is high interest in these reactions for biotechnology and pharmaceutical processes. Many P450s require membrane surroundings and have substrates that do not cross biological membranes. To circumvent these obstacles, CYP106A2 from Bacillus megaterium was expressed on the outer membrane of Escherichia coli cells by Autodisplay. Exposure on the surface was confirmed by a protease accessibility test and flow cytometry after immunolabelling. HPLC assays showed that 0.5 ml of cells displaying the enzyme (OD578 = 6) converted 9.13 µmol of deoxycorticosterone to 15ß-OH-deoxycorticosterone within 1h. Imipramine and abietic acid were also accepted as substrates. The number of active enzyme molecules per cell was calculated to be 20,000. Surprisingly, surface-exposed CYP106A2 was active in E. coli BL21 without the external addition of the heme group. However, when CYP106A2 was expressed on the surface of an E. coli strain lacking the TolC channel protein (JW5503), enzymatic activity was almost completely abolished. The activity of CYP106A2 on the surface of E. coli JW5503 could be restored by the external addition of the heme group. This suggests, as has been reported before, that E. coli uses a TolC-dependent mechanism to export heme into the growth media, where it can be scavenged by a surface-displayed apoenzyme. Our results indicate that Autodisplay enables the functional surface display of P450 enzymes and provides a new platform to access their synthetic potential.

Overexpressed esterases in a fenvalerate resistant strain of the cotton bollworm, Helicoverpa armigera.

Enhanced detoxification is the major mechanism responsible for pyrethroid resistance in Chinese populations of Helicoverpa armigera. Previous work has shown that enhanced oxidation contributes to resistance in the fenvalerate-selected Chinese strain, YGF. The current study provides evidence that enhanced hydrolysis by esterase isozymes also contributes to the resistance in this strain. The average esterase activity of third instar YGF larvae was 1.9-fold compared with that of a susceptible SCD strain. Much of this difference was attributed to isozymes at two zones which hydrolysed the model carboxylester substrate 1-naphthyl acetate and also a 1-naphthyl analogue of fenvalerate. A preparation enriched for enzymes migrating to one of these zones from YGF was shown to hydrolyse fenvalerate with a specific activity of about 2.9 nmol/min/mg. This material was also matched by mass spectrometry with four putative carboxylesterase genes, all of which clustered within a phylogenetic clade of secreted midgut esterases. Quantitative PCR on these four genes showed several-fold greater expression in tissues of YGF compared to SCD but no differences was found in the number of copies of the genes between the strains.

Gene identification and proteomic analysis of the esterases of the cotton bollworm, Helicoverpa armigera.

Some of the resistance of Helicoverpa armigera to conventional insecticides such as organophosphates and synthetic pyrethroids appears to be due to metabolic detoxification by carboxylesterases. To investigate the H. armigera carboxyl/cholinesterases, we created a data set of 39 putative paralogous H. armigera carboxyl/cholinesterase sequences from cDNA libraries and other sources. Phylogenetic analysis revealed a close relationship between these sequences and 70 carboxyl/cholinesterases from the recently sequenced genome of the silkworm, Bombyx mori, including several conserved clades of non-catalytic proteins. A juvenile hormone esterase candidate from H. armigera was identified, and B. mori orthologues were proposed for 31% of the sequences examined, however low similarity was found between lepidopteran sequences and esterases previously associated with insecticide resistance from other insect orders. A proteomic analysis of larval esterases then enabled us to match seven of the H. armigera carboxyl/cholinesterase sequences to specific esterase isozymes. All identified sequences were predicted to encode catalytically active carboxylesterases, including six proteins with N-terminal signal peptides and N-glycans, with two also containing C-terminal signals for glycosylphosphatidylinositol anchor attachment. Five of these sequences were matched to zones of activity on native PAGE at relative mobility values previously associated with insecticide resistance in this species.