Stepwise Evolution of a Buried Inhibitor Peptide over 45 My.

The de novo evolution of genes and the novel proteins they encode has stimulated much interest in the contribution such innovations make to the diversity of life. Most research on this de novo evolution focuses on transcripts, so studies on the biochemical steps that can enable completely new proteins to evolve and the time required to do so have been lacking. Sunflower Preproalbumin with SFTI-1 (PawS1) is an unusual albumin precursor because in addition to producing albumin it also yields a potent, bicyclic protease-inhibitor called SunFlower Trypsin Inhibitor-1 (SFTI-1). Here, we show how this inhibitor peptide evolved stepwise over tens of millions of years. To trace the origin of the inhibitor peptide SFTI-1, we assembled seed transcriptomes for 110 sunflower relatives whose evolution could be resolved by a chronogram, which allowed dates to be estimated for the various stages of molecular evolution. A genetic insertion event in an albumin precursor gene ∼45 Ma introduced two additional cleavage sites for protein maturation and conferred duality upon PawS1-Like genes such that they also encode a small buried macrocycle. Expansion of this region, including two Cys residues, enlarged the peptide ∼34 Ma and made the buried peptides bicyclic. Functional specialization into a protease inhibitor occurred ∼23 Ma. These findings document the evolution of a novel peptide inside a benign region of a pre-existing protein. We illustrate how a novel peptide can evolve without de novo gene evolution and, critically, without affecting the function of what becomes the protein host.

The design and structural characterization of a synthetic pentatricopeptide repeat protein.

Proteins of the pentatricopeptide repeat (PPR) superfamily are characterized by tandem arrays of a degenerate 35-amino-acid α-hairpin motif. PPR proteins are typically single-stranded RNA-binding proteins with essential roles in organelle biogenesis, RNA editing and mRNA maturation. A modular, predictable code for sequence-specific binding of RNA by PPR proteins has recently been revealed, which opens the door to the de novo design of bespoke proteins with specific RNA targets, with widespread biotechnological potential. Here, the design and production of a synthetic PPR protein based on a consensus sequence and the determination of its crystal structure to 2.2 Šresolution are described. The crystal structure displays helical disorder, resulting in electron density representing an infinite superhelical PPR protein. A structural comparison with related tetratricopeptide repeat (TPR) proteins, and with native PPR proteins, reveals key roles for conserved residues in directing the structure and function of PPR proteins. The designed proteins have high solubility and thermal stability, and can form long tracts of PPR repeats. Thus, consensus-sequence synthetic PPR proteins could provide a suitable backbone for the design of bespoke RNA-binding proteins with the potential for high specificity.

Directed evolution of nitrobenzene dioxygenase for the synthesis of the antioxidant hydroxytyrosol.

Nitrobenzene dioxygenase (NBDO) is known to add both atoms of molecular oxygen to the aromatic ring of nitrobenzene to form catechol. It is assembled by four subunits of which the alpha subunit is responsible for catalysis. As an oxidizing enzyme, it has a potential use in the detoxification of industrial waste and the synthesis of pharmaceuticals and food ingredients; however, not much work has been done studying its structure-function correlations. We used several protein engineering approaches (neutral drift libraries, random libraries, two types of focused libraries, and family shuffling) to engineer NBDO for the production of the highly potent antioxidant, hydroxytyrosol (HTyr), from the substrate 3-nitrophenethyl alcohol (3NPA). We obtained a triple mutant, F222C/F251L/G253D, which is able to oxidize 3NPA 375-fold better than wild type with a very high regioselectivity. In total, we identified four positions which are important for acquisition of new specificities, of which only one is well-known and studied. Based on homology modeling, it is suggested that these mutations increase activity by vacating extra space within the active site for the larger substrate and also by hydrogen bonding to the substrate. The best variant had acquired a stabilizing mutation which was beneficial only in this mutant. Thus, we have achieved two goals, the first is the enzymatic production of HTyr, and the second is valuable information regarding the structure-function correlations of NBDO.

Cofactor-independent RNA editing by a synthetic S-type PPR protein.

Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that are attractive tools for RNA processing in synthetic biology applications given their modular structure and ease of design. Several distinct types of motifs have been described from natural PPR proteins, but almost all work so far with synthetic PPR proteins has focused on the most widespread P-type motifs. We have investigated synthetic PPR proteins based on tandem repeats of the more compact S-type PPR motif found in plant organellar RNA editing factors and particularly prevalent in the lycophyte Selaginella. With the aid of a novel plate-based screening method, we show that synthetic S-type PPR proteins are easy to design and bind with high affinity and specificity and are functional in a wide range of pH, salt and temperature conditions. We find that they outperform a synthetic P-type PPR scaffold in many situations. We designed an S-type editing factor to edit an RNA target in E. coli and demonstrate that it edits effectively without requiring any additional cofactors to be added to the system. These qualities make S-type PPR scaffolds ideal for developing new RNA processing tools.

The genetic basis of cytoplasmic male sterility and fertility restoration in wheat.

Hybrid wheat varieties give higher yields than conventional lines but are difficult to produce due to a lack of effective control of male fertility in breeding lines. One promising system involves the Rf1 and Rf3 genes that restore fertility of wheat plants carrying Triticum timopheevii-type cytoplasmic male sterility (T-CMS). Here, by genetic mapping and comparative sequence analyses, we identify Rf1 and Rf3 candidates that can restore normal pollen production in transgenic wheat plants carrying T-CMS. We show that Rf1 and Rf3 bind to the mitochondrial orf279 transcript and induce cleavage, preventing expression of the CMS trait. The identification of restorer genes in wheat is an important step towards the development of hybrid wheat varieties based on a CMS-Rf system. The characterisation of their mode of action brings insights into the molecular basis of CMS and fertility restoration in plants.

Peptide macrocyclization by a bifunctional endoprotease.

Proteases usually cleave peptides, but under some conditions, they can ligate them. Seeds of the common sunflower contain the 14-residue, backbone-macrocyclic peptide sunflower trypsin inhibitor 1 (SFTI-1) whose maturation from its precursor has a genetic requirement for asparaginyl endopeptidase (AEP). To provide more direct evidence, we developed an in situ assay and used (18)O-water to demonstrate that SFTI-1 is excised and simultaneously macrocyclized from its linear precursor. The reaction is inefficient in situ, but a newfound breakdown pathway can mask this inefficiency by reducing the internal disulfide bridge of any acyclic-SFTI to thiols before degrading it. To confirm AEP can directly perform the excision/ligation, we produced several recombinant plant AEPs in E. coli, and one from jack bean could catalyze both a typical cleavage reaction and cleavage-dependent, intramolecular transpeptidation to create SFTI-1. We propose that the evolution of ligating endoproteases enables plants like sunflower and jack bean to stabilize bioactive peptides.

Next generation sequencing and de novo transcriptomics to study gene evolution.

BACKGROUND: Studying gene evolution in non-model species by PCR-based approaches is limited to highly conserved genes. The plummeting cost of next generation sequencing enables the application of de novo transcriptomics to any species. RESULTS: Here we describe how to apply de novo transcriptomics to pursue the evolution of a single gene of interest. We follow a rapidly evolving seed protein that encodes small, stable peptides. We use software that needs limited bioinformatics background and assemble four de novo seed transcriptomes. To demonstrate the quality of the assemblies, we confirm the predicted genes at the peptide level on one species which has over ten copies of our gene of interest. We explain strategies that favour assembly of low abundance genes, what assembly parameters help capture the maximum number of transcripts, how to develop a suite of control genes to test assembly quality and we compare several sequence depths to optimise cost and data volume. CONCLUSIONS: De novo transcriptomics is an effective approach for studying gene evolution in species for which genome support is lacking.

Protein engineering of nirobenzene dioxygenase for enantioselective synthesis of chiral sulfoxides.

Nitrobenzene dioxygenase (NBDO) from Comamonas sp. is shown here to perform enantioselective oxidation of aromatic sulfides. Several para-substituted alkyl aryl sulfides were examined and it was found that the activity of the enzyme is dependent on the size of the substrate. Saturation mutagenesis was performed on different residues in the active site in order to improve activity and selectivity. Mutagenesis at position 258 in the α-hydroxylase subunit of NBDO improved both activity and enantioselectivity. Substitutions in position 293 improved the activity on all substrates and had diverse influence on enantioselectivity. Mutagenesis in position 207 provided two interesting variants, V207I and V207A, with opposite enantioselectivities. Furthermore, combining two favorable mutations, N258A and F293H, provided an improved variant with both higher activity (5.20 ± 0.01, 2.12 ± 0.21, 2.64 ± 0.14 and 4.01 ± 0.34 nmol min(-1) mg protein(-1) on thioanisole, ptolyl, Cl-thioanisole and Br-thioanisole, respectively, which is 1.7, 4.6, 7.1 and 26.7-fold compared with wild type) and improved enantioselectivity (e.g. 67% enantiomeric excess for Cl-thioanisole vs. 5% for wild type). Molecular docking and active site volume calculations were used to correlate between the structure of the substrates and the function of the enzymes. The results from this work suggest that the location of pro-chiral sulfides in the active site is coordinated by hydrophobic interactions and by steric considerations, which in turn influences the activity and enantioselectivity of NBDO.