Converting a Natural Protein Compartment into a Nanofactory for the Size-Constrained Synthesis of Antimicrobial Silver Nanoparticles

Engineered biological systems are used extensively for the production of high value and commodity organics. On the other hand, most inorganic nanomaterials are still synthesized <i>via</i> chemical routes. By engineering cellular compartments, functional nanoarchitectures can be produced under environmentally sustainable conditions. Encapsulins are a new class of microbial nanocompartments with promising applications in nanobiotechnology. Here, we engineer the <i>Thermotoga maritima</i> encapsulin EncTm to yield a designed compartment for the size-constrained synthesis of silver nanoparticles (Ag NPs). These Ag NPs exhibit uniform shape and size distributions as well as long-term stability. Ambient aqueous conditions can be used for Ag NP synthesis, while no reducing agents or solvents need to be added. The antimicrobial activity of the synthesized protein-coated or shell-free Ag NPs is superior to that of silver nitrate and citrate-capped Ag NPs. This study establishes encapsulins as an engineerable platform for the synthesis of biogenic functional nanomaterials

An Enzymatic Pathway for the Biosynthesis of the Formylhydroxyornithine Required for Rhodochelin Iron Coordination

Rhodochelin, a mixed catecholate–hydroxamate type siderophore isolated from <i>Rhodococcus jostii</i> RHA1, holds two l-δ-<i>N</i>-formyl-δ-<i>N</i>-hydroxyornithine (l-fhOrn) moieties essential for proper iron coordination. Previously, bioinformatic and genetic analysis proposed <i>rmo</i> and <i>rft</i> as the genes required for the tailoring of the l-ornithine (l-Orn) precursor [Bosello, M. (2011) <i>J. Am. Chem. Soc.</i> <i>133</i>, 4587–4595]. In order to investigate if both Rmo and Rft constitute a pathway for l-fhOrn biosynthesis, the enzymes were heterologously produced and assayed <i>in vitro</i>. In the presence of molecular oxygen, NADPH and FAD, Rmo monooxygenase was able to convert l-Orn into l-δ-<i>N</i>-hydroxyornithine (l-hOrn). As confirmed in a coupled reaction assay, this hydroxylated intermediate serves as a substrate for the subsequent <i>N</i>¹⁰-formyl-tetrahydrofolate-dependent (<i>N</i>¹⁰-fH₄F) Rtf-catalyzed formylation reaction, establishing a route for the l-fhOrn biosynthesis, prior to its incorporation by the NRPS assembly line. It is of particular interest that a major improvement to this study has been reached with the use of an alternative approach to the chemoenzymatic FolD-dependent <i>N</i>¹⁰-fH₄F conversion, also rescuing the previously inactive CchA, the Rft-homologue in coelichelin assembly line [Buchenau, B. (2004) <i>Arch. Microbiol.</i> <i>182</i>, 313–325; Pohlmann, V. (2008) <i>Org. Biomol. Chem.</i> <i>6</i>, 1843–1848]

Two [4Fe-4S] Clusters Containing Radical SAM Enzyme SkfB Catalyze Thioether Bond Formation during the Maturation of the Sporulation Killing Factor

The sporulation killing factor (SKF) is a 26-residue ribosomally assembled and posttranslationally modified sactipeptide. It is produced by <i>Bacillus subtilis</i> 168 and plays a key role in its sporulation. Like all sactipeptides, SKF contains a thioether bond, which links the cysteine residue Cys4 with the α-carbon of the methionine residue Met12. In this study we demonstrate that this bond is generated by the two [4Fe-4S] clusters containing radical SAM enzyme SkfB, which is encoded in the <i>skf</i> operon. By mutational analysis of both cluster-binding sites, we were able to postulate a mechanism for thioether generation which is in agreement with that of AlbA. Furthermore, we were able to show that thioether bond formation is specific toward hydrophobic amino acids at the acceptor site. Additionally we demonstrate that generation of the thioether linkage is leader-peptide-dependent, suggesting that this reaction is the first step in SKF maturation