Association of Radical Chemistry with LanD Flavoprotein Activity for C-Terminal Macrocyclization of a Ribosomal Peptide by Formation of an Unsaturated Thioether Residue.

LanD flavoproteins catalyze oxidative decarboxylation of the C-terminal Cys residue of a peptide to produce an enethiol. This enethiol is highly reactive and can be coupled with an upstream dehydroamino acid through Michael addition to form S-[2-aminovinyl](3-methyl)cysteine, an unsaturated thioether residue known to be characteristic of an array of C-terminally macrocyclized, ribosomally synthesized and posttranslationally modified peptides (RiPPs). Based on a two-stage bioinformatics mining of posttranslational modifications (PTMs) related to C-terminal Cys processing, we report herein that LanD activity can couple with radical S-adenosylmethionine chemistry to provide a new unsaturated thioether residue, S-[2-aminovinyl]-3-carbamoylcysteine, by conjugating the resultant enethiol with Cß of the Asn residue in the C-terminal NxxC motif of a peptide for macrocyclization. This study furthers our understanding of the variety of PTMs involved in creating the structure diversity of macrocyclic RiPPs.

Dissection of the Enzymatic Process for Forming a Central Imidazopiperidine Heterocycle in the Biosynthesis of a Series c Thiopeptide Antibiotic.

Thiopeptide antibiotics are a family of ribosomally synthesized and posttranslationally modified peptide natural products of significant interest in anti-infective agent development. These antibiotics are classified into five subfamilies according to differences in the central 6-membered heterocycle of the thiopeptide framework. The mechanism through which imidazopiperidine, the most heavily functionalized central domain characteristic of a series c thiopeptide, is formed remains unclear. Based on mining and characterization of the genes specifically involved in the biosynthesis of Sch40832, we here report an enzymatic process for transforming a series b thiopeptide into a series c product through a series a intermediate. This process starts with F420-dependent hydrogenation of the central dehydropiperidine unit to a saturated piperidine unit. With the activity of a cytochrome P450 monooxygenase, the piperidine-thiazole motif of the intermediate undergoes an unusual oxygenation-mediated rearrangement to provide an imidazopiperidine heterocycle subjected to further S-methylation and aldehyde reduction. This study represents the first biochemical reconstitution of the pathway forming a stable series c thiopeptide.

Enzymatic Formation of an Aminovinyl Cysteine Residue in Ribosomal Peptide Natural Products.

The carboxyl-terminal (C-terminal) S-[(Z)-2-aminovinyl]-cysteine (AviCys) analogs have been identified in four families of ribosomally synthesized and post-translationally modified peptides (RiPPs): lanthipeptides, linaridins, thioamitides, and lipolanthines. Within identified biosynthetic pathways, a highly reactive enethiol intermediate, formed through an oxidative decarboxylation catalyzed by a LanD-like flavoprotein, can undergo two types of cyclization: a Michael addition with a dehydroamino acid or a coupling reaction initiated by a radical species. The collaborative actions of LanD-like proteins with diverse enzymes involved in dehydration, dethiolation or cyclization lead to the construction of structurally distinct peptide natural products with analogous C-terminal macrocyclic moieties. This concept summarizes existing knowledge regarding biosynthetic pathways of AviCys analogs to emphasize the diversity of biosynthetic mechanisms that paves the way for future genome mining explorations into diverse peptide natural products.

New insight into the adsorption behaviour of effluent organic matter on organic-inorganic ultrafiltration membranes: a combined QCM-D and AFM study.

Adsorption of organic matter on membranes plays a major role in determining the fouling behaviour of membranes. This study investigated effluent organic matter (EfOM) adsorption behaviour onto poly(vinylidene fluoride) (PVDF) membrane blended with SiO2 nanoparticles using quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). The QCM-D results suggested that low adsorption of EfOM and an EfOM layer with a non-rigid and open structure was formed on SiO2-terminated membrane surfaces. Conformational assessment showed that EfOM undergoes adsorption via two steps: (i) in the initial stage, a rapid adsorption of EfOM accumulated onto the membrane; (ii) the change in dissipation was still occurring when the adsorption frequency reached balance, and the layer tended towards a more rearranged or organized secondary structure upon adsorption onto the more hydrophilic surface. For the AFM force test, when a self-made EfOM-coated probe approached the membrane, a 'jump-in' was observed for the hydrophobic membrane after repulsion at a small distance, while only repulsive forces were observed for PVDF/SiO2 membranes. This study demonstrated that the PVDF/SiO2 membrane changed the entire filtration process, forming a 'soft' open conformation in the foulant layer.

Effects of hydraulic retention time on adsorption behaviours of EPS in an A/O-MBR: biofouling study with QCM-D.

Extra-cellular polymeric substances (EPS) are a major cause of membrane fouling in membrane bioreactors (MBRs). In this study, an anoxic-oxic membrane bioreactor (A/O-MBR) was run continuously for 98 days. The runs were divided into three stages according to hydraulic retention time (HRT) (11.8, 12.5 and 14.3 h, respectively). EPS were extracted from the reactor under the different HRTs. A quartz crystal microbalance with dissipation monitoring (QCM-D) and Fourier transform infrared (FT-IR) were used to study the adherence layer structures and the adsorption behaviours of EPS on the membrane surface. The results indicated that the removal rate of TN was more susceptible to HRT than NH3-N. The observations in the QCM-D suggested that at the lowest HRT (11.8 h), the structure of the adsorption layer is loose and soft and the fluidity was better than for HRTs of 12.5 or 14.3 h. It is likely one of the major reasons for the rapidly blocking of the membrane pores. Furthermore, the higher EPS adherence as analyzed in the QCM-D and EPS concentration could induce a higher osmotic pressure effect, leading to a rapid membrane-fouling rate.