Discovery and Characterization of Rubrinodin Provide Clues into the Evolution of Lasso Peptides.

Lasso peptides are unique natural products that comprise a class of ribosomally synthesized and post-translationally modified peptides. Their defining three-dimensional structure is a lariat knot, in which the C-terminal tail is threaded through a macrolactam ring formed between the N-terminal amino group and an Asp or Glu side chain (i.e., an isopeptide bond). Recent genome mining strategies have revealed various types of lasso peptide biosynthetic gene clusters and have thus redefined the known chemical space of lasso peptides. To date, over 20 different types of these gene clusters have been discovered, including several different clades from Proteobacteria. Despite the diverse architectures of these gene clusters, which may or may not encode various tailoring enzymes, most currently known lasso peptides are synthesized by two discrete clades defined by the presence of an ATP-binding cassette transporter or its absence and (sometimes) concurrent appearance of an isopeptidase, raising questions about their evolutionary history. Herein, we discovered and characterized the lasso peptide rubrinodin, which is assembled by a gene cluster encoding both an ATP-binding cassette transporter and an isopeptidase. Our bioinformatics analyses of this and other representative cluster types provided new clues into the evolutionary history of lasso peptides. Furthermore, our structural and biochemical investigations of rubrinodin permitted the conversion of this thermolabile lasso peptide into a more thermostable scaffold.

Structural and mutational analysis of the nonribosomal peptide synthetase heterocyclization domain provides insight into catalysis.

Nonribosomal peptide synthetases (NRPSs) are a family of multidomain, multimodule enzymes that synthesize structurally and functionally diverse peptides, many of which are of great therapeutic or commercial value. The central chemical step of peptide synthesis is amide bond formation, which is typically catalyzed by the condensation (C) domain. In many NRPS modules, the C domain is replaced by the heterocyclization (Cy) domain, a homologous domain that performs two consecutive reactions by using hitherto unknown catalytic mechanisms. It first catalyzes amide bond formation, and then the intramolecular cyclodehydration between a Cys, Ser, or Thr side chain and the backbone carbonyl carbon to form a thiazoline, oxazoline, or methyloxazoline ring. The rings are important for the form and function of the peptide product. We present the crystal structure of an NRPS Cy domain, Cy2 of bacillamide synthetase, at a resolution of 2.3 Å. Despite sharing the same fold, the active sites of C and Cy domains have important differences. The structure allowed us to probe the roles of active-site residues by using mutational analyses in a peptide synthesis assay with intact bacillamide synthetase. The drastically different effects of these mutants, interpreted by using our structural and bioinformatic results, provide insight into the catalytic mechanisms of the Cy domain and implicate a previously unexamined Asp-Thr dyad in catalysis of the cyclodehydration reaction.

The Kalimantacin Polyketide Antibiotics Inhibit Fatty Acid Biosynthesis in Staphylococcus aureus by Targeting the Enoyl-Acyl Carrier Protein Binding Site of FabI.

The enoyl-acyl carrier protein reductase enzyme FabI is essential for fatty acid biosynthesis in Staphylococcus aureus and represents a promising target for the development of novel, urgently needed anti-staphylococcal agents. Here, we elucidate the mode of action of the kalimantacin antibiotics, a novel class of FabI inhibitors with clinically-relevant activity against multidrug-resistant S. aureus. By combining X-ray crystallography with molecular dynamics simulations, in vitro kinetic studies and chemical derivatization experiments, we characterize the interaction between the antibiotics and their target, and we demonstrate that the kalimantacins bind in a unique conformation that differs significantly from the binding mode of other known FabI inhibitors. We also investigate mechanisms of acquired resistance in S. aureus and identify key residues in FabI that stabilize the binding of the antibiotics. Our findings provide intriguing insights into the mode of action of a novel class of FabI inhibitors that will inspire future anti-staphylococcal drug development.

Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone.

Nucleotide-based second messengers serve in the response of living organisms to environmental changes. In bacteria and plant chloroplasts, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named "(p)ppGpp"] act as alarmones that globally reprogram cellular physiology during various stress conditions. Enzymes of the RelA/SpoT homology (RSH) family synthesize (p)ppGpp by transferring pyrophosphate from ATP to GDP or GTP. Little is known about the catalytic mechanism and regulation of alarmone synthesis. It also is unclear whether ppGpp and pppGpp execute different functions. Here, we unravel the mechanism and allosteric regulation of the highly cooperative alarmone synthetase small alarmone synthetase 1 (SAS1) from Bacillus subtilis. We determine that the catalytic pathway of (p)ppGpp synthesis involves a sequentially ordered substrate binding, activation of ATP in a strained conformation, and transfer of pyrophosphate through a nucleophilic substitution (SN2) reaction. We show that pppGpp-but not ppGpp-positively regulates SAS1 at an allosteric site. Although the physiological significance remains to be elucidated, we establish the structural and mechanistic basis for a biological activity in which ppGpp and pppGpp execute different functional roles.

Insights into the Unique Phosphorylation of the Lasso Peptide Paeninodin.

Lasso peptides are a new class of ribosomally synthesized and post-translationally modified peptides and thus far are only isolated from proteo- and actinobacterial sources. Typically, lasso peptide biosynthetic gene clusters encode enzymes for biosynthesis and export but not for tailoring. Here, we describe the isolation of the novel lasso peptide paeninodin from the firmicute Paenibacillus dendritiformis C454 and reveal within its biosynthetic cluster a gene encoding a kinase, which we have characterized as a member of a new class of lasso peptide-tailoring kinases. By employing a wide variety of peptide substrates, it was shown that this novel type of kinase specifically phosphorylates the C-terminal serine residue while ignoring those located elsewhere. These experiments also reveal that no other recognition motif is needed for efficient enzymatic phosphorylation of the C-terminal serine. Furthermore, through comparison with homologous HPr kinases and subsequent mutational analysis, we confirmed the essential catalytic residues. Our study reveals how lasso peptides are chemically diversified and sets the foundation for rational engineering of these intriguing natural products.

The structure of SpnF, a standalone enzyme that catalyzes [4 + 2] cycloaddition.

In the biosynthetic pathway of the spinosyn insecticides, the tailoring enzyme SpnF performs a [4 + 2] cycloaddition on a 22-membered macrolactone to forge an embedded cyclohexene ring. To learn more about this reaction, which could potentially proceed through a Diels-Alder mechanism, we determined the 1.50-Å-resolution crystal structure of SpnF bound to S-adenosylhomocysteine. This sets the stage for advanced experimental and computational studies to determine the precise mechanism of SpnF-mediated cyclization.

Structure and Mechanism of the Sphingopyxin†I Lasso Peptide Isopeptidase.

Lasso peptides are natural products that assume a unique lariat knot topology. Lasso peptide isopeptidases (IsoPs) eliminate this topology through isopeptide bond cleavage. To probe how these enzymes distinguish between substrates and hydrolyze only isopeptide bonds, we examined the structure and mechanism of a previously uncharacterized IsoP from the proteobacterium Sphingopyxis alaskensis RB2256 (SpI-IsoP). We demonstrate that SpI-IsoP efficiently and specifically linearizes the lasso peptide sphingopyxin I (SpI) and variants thereof. We also present crystal structures of SpI and SpI-IsoP, revealing a threaded topology for the former and a prolyl oligopeptidase (POP)-like fold for the latter. Subsequent structure-guided mutational analysis allowed us to propose roles for active-site residues. Our study sheds light on lasso peptide catabolism and expands the engineering potential of these fascinating molecules.

Coenzyme A-free activity, crystal structure, and rational engineering of a promiscuous ß-ketoacyl thiolase from Ralstonia eutropha.

Thiolases catalyze the formation of carbon-carbon bonds in diverse biosynthetic pathways. The promiscuous ß-ketoacyl thiolase B of Ralstonia eutropha (ReBktB) has been utilized in the in vivo conversion of Coenzyme A (CoA)-linked precursors such as acetyl-CoA and glycolyl-CoA into ß-hydroxy acids, including the pharmaceutically-important 3,4-dihydroxybutyric acid. Such thiolases could serve as powerful carbon-carbon bond-forming biocatalysts in vitro if handles less costly than CoA were employable. Here, thiolase activity is demonstrated toward substrates linked to the readily-available CoA mimic, N-acetylcysteamine (NAC). ReBktB was observed to catalyze the retro-Claisen condensation of several ß-ketoacyl-S-NAC substrates, with a preference for 3-oxopentanoyl-S-NAC over 3-oxobutanoyl-, 3-oxohexanoyl-, and 3-oxoheptanoyl-S-NAC. A 2.0 Å-resolution crystal structure, in which the asymmetric unit consists of four ReBktB tetramers, provides insight into acyl group specificity and how it may be engineered. By replacing an active site methionine with an alanine, a mutant possessing significant activity towards α-methyl substituted, NAC-linked substrates was engineered. The ability of ReBktB and its engineered mutants to utilize NAC-linked substrates will facilitate the in vitro biocatalytic synthesis of diketide chiral building blocks from feedstock molecules such as acetate and propionate.

Crystallographic study of the phosphoethanolamine transferase EptC required for polymyxin resistance and motility in Campylobacter jejuni.

The foodborne enteric pathogen Campylobacter jejuni decorates a variety of its cell-surface structures with phosphoethanolamine (pEtN). Modifying lipid A with pEtN promotes cationic antimicrobial peptide resistance, whereas post-translationally modifying the flagellar rod protein FlgG with pEtN promotes flagellar assembly and motility, which are processes that are important for intestinal colonization. EptC, the pEtN transferase required for all known pEtN cell-surface modifications in C. jejuni, is a predicted inner-membrane metalloenzyme with a five-helix N-terminal transmembrane domain followed by a soluble sulfatase-like catalytic domain in the periplasm. The atomic structure of the catalytic domain of EptC (cEptC) was crystallized and solved to a resolution of 2.40 Å. cEptC adopts the α/ß/α fold of the sulfatase protein family and harbors a zinc-binding site. A phosphorylated Thr266 residue was observed that was hypothesized to mimic a covalent pEtN-enzyme intermediate. The requirement for Thr266 as well as the nearby residues Asn308, Ser309, His358 and His440 was ascertained via in vivo activity assays on mutant strains. The results establish a basis for the design of pEtN transferase inhibitors.

Native ESI-MS and Collision-Induced Unfolding (CIU) of the Complex between Bacterial Elongation Factor-Tu and the Antibiotic Enacyloxin IIa.

Collision-induced unfolding (CIU) of protein ions, monitored by ion mobility-mass spectrometry, can be used to assess the stability of their compact gas-phase fold and hence provide structural information. The bacterial elongation factor EF-Tu, a key protein for mRNA translation in prokaryotes and hence a promising antibiotic target, has been studied by CIU. The major [M + 12H]12+ ion of EF-Tu unfolded in collision with Ar atoms between 40 and 50 V, corresponding to an Elab energy of 480-500 eV. Binding of the cofactor analogue GDPNP and the antibiotic enacyloxin IIa stabilized the compact fold of EF-Tu, although dissociation of the latter from the complex diminished its stabilizing effect at higher collision energies. Molecular dynamics simulations of the [M + 12H]12+ EF-Tu ion showed similar qualitative behavior to the experimental results.