Exploring the Post-translational Enzymology of PaaA by mRNA Display.

PaaA is a RiPP enzyme that catalyzes the transformation of two glutamic acid residues within a substrate peptide into the bicyclic core of Pantocin A. Here, for the first time, we use mRNA display techniques to understand RiPP enzyme-substrate interactions to illuminate PaaA substrate recognition. Additionally, our data revealed insights into the enzymatic timing of glutamic acid modification. The technique developed is quite sensitive and a significant advancement over current RiPP studies and opens the door to enzyme modified mRNA display libraries for natural product-like inhibitor pans.

Structural Insights into Thioether Bond Formation in the Biosynthesis of Sactipeptides.

Sactipeptides are ribosomally synthesized peptides that contain a characteristic thioether bridge (sactionine bond) that is installed posttranslationally and is absolutely required for their antibiotic activity. Sactipeptide biosynthesis requires a unique family of radical SAM enzymes, which contain multiple [4Fe-4S] clusters, to form the requisite thioether bridge between a cysteine and the α-carbon of an opposing amino acid through radical-based chemistry. Here we present the structure of the sactionine bond-forming enzyme CteB, from Clostridium thermocellum ATCC 27405, with both SAM and an N-terminal fragment of its peptidyl-substrate at 2.04 Å resolution. CteB has the (ß/α)6-TIM barrel fold that is characteristic of radical SAM enzymes, as well as a C-terminal SPASM domain that contains two auxiliary [4Fe-4S] clusters. Importantly, one [4Fe-4S] cluster in the SPASM domain exhibits an open coordination site in absence of peptide substrate, which is coordinated by a peptidyl-cysteine residue in the bound state. The crystal structure of CteB also reveals an accessory N-terminal domain that has high structural similarity to a recently discovered motif present in several enzymes that act on ribosomally synthesized and post-translationally modified peptides (RiPPs), known as a RiPP precursor peptide recognition element (RRE). This crystal structure is the first of a sactionine bond forming enzyme and sheds light on structures and mechanisms of other members of this class such as AlbA or ThnB.

Production of Sactipeptides in Escherichia coli: Probing the Substrate Promiscuity of Subtilosin A Biosynthesis.

Sactipeptides are peptide-derived natural products that are processed by remarkable, radical-mediated cysteine sulfur to α-carbon coupling reactions. The resulting sactionine thioether linkages give rise to the unique defined structures and concomitant biological activities of sactipeptides. An E. coli heterologous expression system, based on the biosynthesis of one such sactipeptide, subtilosin A, is described and this expression system is exploited to probe the promiscuity of the subtilosin A sactionine bond-forming enzyme, AlbA. These efforts allowed the facile expression and isolation of a small library of mutant sactipeptides based on the subtilosin A precursor peptide, demonstrating broad substrate promiscuity where none was previously known. Importantly, we show that the positions of the sactionine linkages can be moved, giving rise to new, unnatural sactipeptide structures. E. coli heterologous expression also allowed incorporation of unnatural amino acids into sactipeptides by means of amber-suppression technology, potentially opening up new chemistry and new applications for unnatural sactipeptides.

Pheromone killing of multidrug-resistant Enterococcus faecalis V583 by native commensal strains.

Multidrug-resistant Enterococcus faecalis possess numerous mobile elements that encode virulence and antibiotic resistance traits as well as new metabolic pathways, often constituting over one-quarter of the genome. It was of interest to determine how this large accretion of mobile elements affects competitive growth in the gastrointestinal (GI) tract consortium. We unexpectedly observed that the prototype clinical isolate strain V583 was actively killed by GI tract flora, whereas commensal enterococci flourished. It was found that killing of V583 resulted from lethal cross-talk between accumulated mobile elements and that this cross-talk was induced by a heptapeptide pheromone produced by native E. faecalis present in the fecal consortium. These results highlight two important aspects of the evolution of multidrug-resistant enterococci: (i) the accretion of mobile elements in E. faecalis V583 renders it incompatible with commensal strains, and (ii) because of this incompatibility, multidrug-resistant strains sharing features found in V583 cannot coexist with commensal strains. The accumulation of mobile elements in hospital isolates of enterococci can include those that are inherently incompatible with native flora, highlighting the importance of maintaining commensal populations as means of preventing colonization and subsequent infection by multidrug-resistant strains.

Structure of bacterial transcription factor SpoIIID and evidence for a novel mode of DNA binding.

SpoIIID is evolutionarily conserved in endospore-forming bacteria, and it activates or represses many genes during sporulation of Bacillus subtilis. An SpoIIID monomer binds DNA with high affinity and moderate sequence specificity. In addition to a predicted helix-turn-helix motif, SpoIIID has a C-terminal basic region that contributes to DNA binding. The nuclear magnetic resonance (NMR) solution structure of SpoIIID in complex with DNA revealed that SpoIIID does indeed have a helix-turn-helix domain and that it has a novel C-terminal helical extension. Residues in both of these regions interact with DNA, based on the NMR data and on the effects on DNA binding in vitro of SpoIIID with single-alanine substitutions. These data, as well as sequence conservation in SpoIIID binding sites, were used for information-driven docking to model the SpoIIID-DNA complex. The modeling resulted in a single cluster of models in which the recognition helix of the helix-turn-helix domain interacts with the major groove of DNA, as expected. Interestingly, the C-terminal extension, which includes two helices connected by a kink, interacts with the adjacent minor groove of DNA in the models. This predicted novel mode of binding is proposed to explain how a monomer of SpoIIID achieves high-affinity DNA binding. Since SpoIIID is conserved only in endospore-forming bacteria, which include important pathogenic Bacilli and Clostridia, whose ability to sporulate contributes to their environmental persistence, the interaction of the C-terminal extension of SpoIIID with DNA is a potential target for development of sporulation inhibitors.

Two regions of Bacillus subtilis transcription factor SpoIIID allow a monomer to bind DNA.

Nutrient limitation causes Bacillus subtilis to develop into two different cell types, a mother cell and a spore. SpoIIID is a key regulator of transcription in the mother cell and positively or negatively regulates more than 100 genes, in many cases by binding to the promoter region. SpoIIID was predicted to have a helix-turn-helix motif for sequence-specific DNA binding, and a 10-bp consensus sequence was recognized in binding sites, but some strong binding sites were observed to contain more than one match to the consensus sequence, suggesting that SpoIIID might bind as a dimer or cooperatively as monomers. Here we show that SpoIIID binds with high affinity as a monomer to a single copy of its recognition sequence. Using charge reversal substitutions of residues likely to be exposed on the surface of SpoIIID and assays for transcriptional activation in vivo and for DNA binding in vitro, we identify two regions essential for DNA binding, the putative recognition helix of the predicted helix-turn-helix motif and a basic region near the C terminus. SpoIIID is unusual among prokaryotic DNA-binding proteins with a single helix-turn-helix motif in its ability to bind DNA monomerically with high affinity. We propose that the C-terminal basic region of SpoIIID makes additional contacts with DNA, analogous to the N-terminal arm of eukaryotic homeodomain proteins and the "wings" of winged-helix proteins, but structurally distinct. SpoIIID is highly conserved only among bacteria that form endospores, including several important human pathogens. The need to conserve biosynthetic capacity during endospore formation might have favored the evolution of a small transcription factor capable of high-affinity binding to DNA as a monomer, and this unusual mode of DNA binding could provide a target for drug design.