Thermorubin Biosynthesis Initiated by a Salicylate Synthase Suggests an Unusual Conversion of Phenols to Pyrones.

Thermorubin is a tetracyclic naphthoisocoumarin natural product that demands investigation due to its novel mechanism of bacterial protein synthesis inhibition and its unusual structural features. In this work, we describe the identification of the biosynthetic cluster responsible for thermorubin from the sequenced Laceyella sacchari producer species and its confirmation via heterologous production in Escherichia coli. Based on an in-depth annotation of the cluster, we propose a biosynthetic pathway that accounts for the formation of the unique, nonterminal pyrone. Additionally, the expression and use of salicylate synthase TheO enabled testing of the stability properties of this extremophile-derived enzyme. TheO displayed rapid kinetics and a remarkably robust secondary structure, converting chorismate to salicylate with a KM of 109 ± 12 µM, kcat of 9.17 ± 0.36 min-1, and catalytic efficiency (kcat/KM) of 84 ± 9 nM-1 min-1, and retained significant activity up to 50 °C. These studies serve as the basis for continued biosynthetic investigations and bioinspired synthetic approaches.

Engineering repeat proteins of the immune system.

Limitations associated with immunoglobulins have motivated the search for novel binding scaffolds. Repeat proteins have emerged as one promising class of scaffolds, but often are limited to binding protein and peptide targets. An exception is the repeat proteins of the immune system, which have in recent years served as an inspiration for binding scaffolds which can bind glycans and other classes of biomolecule. Like other repeat proteins, these proteins can be very stable and have a monomeric mode of binding, with elongated and highly variable binding surfaces. The ability to target glycans and glycoproteins fill an important gap in current tools for research and biomedical applications.

Homo- and heteropolymer self-assembly of recombinant trichocytic keratins.

In the past two decades, keratin biomaterials have shown impressive results as scaffolds for tissue engineering, wound healing, and nerve regeneration. In addition to its intrinsic biocompatibility, keratin interacts with specific cell receptors eliciting beneficial biochemical cues. However, during extraction from natural sources, such as hair and wool fibers, natural keratins are subject to extensive processing conditions that lead to formation of unwanted by-products. Additionally, natural keratins suffer from limited sequence tunability. Recombinant keratin proteins can overcome these drawbacks while maintaining the desired chemical and physical characteristics of natural keratins. Herein, we present the bacterial expression, purification, and solution characterization of human hair keratins K31 and K81. The obligate heterodimerization of the K31/K81 pair that results in formation of intermediate filaments is maintained in the recombinant proteins. Surprisingly, we have for the first time observed new zero- and one-dimensional nanostructures from homooligomerization of K81 and K31, respectively. Further analysis of the self-assembly mechanism highlights the importance of disulfide crosslinking in keratin self-assembly.