Not always an innocent bystander: the impact of stabilised phosphopantetheine moieties when studying nonribosomal peptide biosynthesis.

Nonribosomal peptide synthetases produce many important peptide natural products and are centred around carrier proteins (CPs) that deliver intermediates to various catalytic domains. We show that the replacement of CP substrate thioesters by stabilised ester analogues leads to active condensation domain complexes, whereas amide stabilisation generates non-functional complexes.

The Cytochrome P450 OxyA from the Kistamicin Biosynthesis Cyclization Cascade is Highly Sensitive to Oxidative Damage.

Cytochrome P450 enzymes (P450s) are a superfamily of monooxygenases that utilize a cysteine thiolate-ligated heme moiety to perform a wide range of demanding oxidative transformations. Given the oxidative power of the active intermediate formed within P450s during their active cycle, it is remarkable that these enzymes can avoid auto-oxidation and retain the axial cysteine ligand in the deprotonated-and thus highly acidic-thiolate form. While little is known about the process of heme incorporation during P450 folding, there is an overwhelming preference for one heme orientation within the P450 active site. Indeed, very few structures to date contain an alternate heme orientation, of which two are OxyA homologs from glycopeptide antibiotic (GPA) biosynthesis. Given the apparent preference for the unusual heme orientation shown by OxyA enzymes, we investigated the OxyA homolog from kistamicin biosynthesis (OxyAkis), which is an atypical GPA. We determined that OxyAkis is highly sensitive to oxidative damage by peroxide, with both UV and EPR measurements showing rapid bleaching of the heme signal. We determined the structure of OxyAkis and found a mixed population of heme orientations present in this enzyme. Our analysis further revealed the possible modification of the heme moiety, which was only present in samples where the alternate heme orientation was present in the protein. These results suggest that the typical heme orientation in cytochrome P450s can help prevent potential damage to the heme-and hence deactivation of the enzyme-during P450 catalysis. It also suggests that some P450 enzymes involved in GPA biosynthesis may be especially prone to oxidative damage due to the heme orientation found in their active sites.

Structures of a non-ribosomal peptide synthetase condensation domain suggest the basis of substrate selectivity.

Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.

Kistamicin biosynthesis reveals the biosynthetic requirements for production of highly crosslinked glycopeptide antibiotics.

Kistamicin is a divergent member of the glycopeptide antibiotics, a structurally complex class of important, clinically relevant antibiotics often used as the last resort against resistant bacteria. The extensively crosslinked structure of these antibiotics that is essential for their activity makes their chemical synthesis highly challenging and limits their production to bacterial fermentation. Kistamicin contains three crosslinks, including an unusual 15-membered A-O-B ring, despite the presence of only two Cytochrome P450 Oxy enzymes thought to catalyse formation of such crosslinks within the biosynthetic gene cluster. In this study, we characterise the kistamicin cyclisation pathway, showing that the two Oxy enzymes are responsible for these crosslinks within kistamicin and that they function through interactions with the X-domain, unique to glycopeptide antibiotic biosynthesis. We also show that the kistamicin OxyC enzyme is a promiscuous biocatalyst, able to install multiple crosslinks into peptides containing phenolic amino acids.

The many faces and important roles of protein-protein interactions during non-ribosomal peptide synthesis.

Covering: up to July 2018 Non-ribosomal peptide synthetase (NRPS) machineries are complex, multi-domain proteins that are responsible for the biosynthesis of many important, peptide-derived compounds. By decoupling peptide synthesis from the ribosome, NRPS assembly lines are able to access a significant pool of amino acid monomers for peptide synthesis. This is combined with a modular protein architecture that allows for great variation in stereochemistry, peptide length, cyclisation state and further modifications. The architecture of NRPS assembly lines relies upon a repetitive set of catalytic domains, which are organised into modules responsible for amino acid incorporation. Central to NRPS-mediated biosynthesis is the carrier protein (CP) domain, to which all intermediates following initial monomer activation are bound during peptide synthesis up until the final handover to the thioesterase domain that cleaves the mature peptide from the NRPS. This mechanism makes understanding the protein-protein interactions that occur between different NRPS domains during peptide biosynthesis of crucial importance to understanding overall NRPS function. This endeavour is also highly challenging due to the inherent flexibility and dynamics of NRPS systems. In this review, we present the current state of understanding of the protein-protein interactions that govern NRPS-mediated biosynthesis, with a focus on insights gained from structural studies relating to CP domain interactions within these impressive peptide assembly lines.

Bacterial actin MreB forms antiparallel double filaments.

Filaments of all actin-like proteins known to date are assembled from pairs of protofilaments that are arranged in a parallel fashion, generating polarity. In this study, we show that the prokaryotic actin homologue MreB forms pairs of protofilaments that adopt an antiparallel arrangement in vitro and in vivo. We provide an atomic view of antiparallel protofilaments of Caulobacter MreB as apparent from crystal structures. We show that a protofilament doublet is essential for MreB's function in cell shape maintenance and demonstrate by in vivo site-specific cross-linking the antiparallel orientation of MreB protofilaments in E. coli. 3D cryo-EM shows that pairs of protofilaments of Caulobacter MreB tightly bind to membranes. Crystal structures of different nucleotide and polymerisation states of Caulobacter MreB reveal conserved conformational changes accompanying antiparallel filament formation. Finally, the antimicrobial agents A22/MP265 are shown to bind close to the bound nucleotide of MreB, presumably preventing nucleotide hydrolysis and destabilising double protofilaments.DOI: http://dx.doi.org/10.7554/eLife.02634.001.

Sortase activity is controlled by a flexible lid in the pilus biogenesis mechanism of gram-positive pathogens.

Pili are surface-linked virulence factors that play key roles in infection establishment in a variety of pathogenic species. In Gram-positive pathogens, pilus formation requires the action of sortases, dedicated transpeptidases that covalently associate pilus building blocks. In Streptococcus pneumoniae, a major human pathogen, all genes required for pilus formation are harbored in a single pathogenicity islet which encodes three structural proteins (RrgA, RrgB, RrgC) and three sortases (SrtC-1, SrtC-2, SrtC-3). RrgB forms the backbone of the streptococcal pilus, to which minor pilins RrgA and RrgC are covalently associated. SrtC-1 is the main sortase involved in polymerization of the RrgB fiber and displays a lid which encapsulates the active site, a feature present in all pilus-related sortases. In this work, we show that catalysis by SrtC-1 proceeds through a catalytic triad constituted of His, Arg, and Cys and that lid instability affects protein fold and catalysis. In addition, we show by thermal shift analysis that lid flexibility can be stabilized by the addition of substrate-like peptides, a feature shared by other periplasmic transpeptidases. We also report the characterization of a trapped acyl-enzyme intermediate formed between SrtC-1 and RrgB. The presence of lid-encapsulated sortases in the pilus biogenesis systems in many Gram-positive pathogens points to a common mechanism of substrate recognition and catalysis that should be taken into consideration in the development of sortase inhibitors.