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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 Author(s) 2022. Published by Oxford University Press.)

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 Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.