Synthetic cycle of the initiation module of a formylating nonribosomal peptide synthetase.

Nonribosomal peptide synthetases (NRPSs) are very large proteins that produce small peptide molecules with wide-ranging biological activities, including environmentally friendly chemicals and many widely used therapeutics. NRPSs are macromolecular machines, with modular assembly-line logic, a complex catalytic cycle, moving parts and many active sites. In addition to the core domains required to link the substrates, they often include specialized tailoring domains, which introduce chemical modifications and allow the product to access a large expanse of chemical space. It is still unknown how the NRPS tailoring domains are structurally accommodated into megaenzymes or how they have adapted to function in nonribosomal peptide synthesis. Here we present a series of crystal structures of the initiation module of an antibiotic-producing NRPS, linear gramicidin synthetase. This module includes the specialized tailoring formylation domain, and states are captured that represent every major step of the assembly-line synthesis in the initiation module. The transitions between conformations are large in scale, with both the peptidyl carrier protein domain and the adenylation subdomain undergoing huge movements to transport substrate between distal active sites. The structures highlight the great versatility of NRPSs, as small domains repurpose and recycle their limited interfaces to interact with their various binding partners. Understanding tailoring domains is important if NRPSs are to be utilized in the production of novel therapeutics.

Evaluation of buccal swabs for pharmacogenetics.

OBJECTIVE: A simple, non-invasive sample collection method is key for the integration of pharmacogenetics into clinical practice. The aim of this study was to gain samples for pharmacogenetic testing and evaluate the variation between dry-flocked and sponge-tipped buccal swabs in yield and quality of DNA isolated. RESULTS: Thirty-one participants collected samples using dry-flocked swabs and sponge-tipped swabs. Samples were assessed for DNA yield, quality and genotyping performance on a qPCR OpenArray platform of 28 pharmacogenetic SNPs and a CYP2D6 TaqMan copy number variant. DNA from sponge-tipped swabs had a significantly greater yield compared to DNA collected with dry-flocked swabs (p = 4.4 × 10-7). Moreover, highest genotyping call rates across all assays and highest CNV confidence scores were observed in DNA samples collected from sponge-tipped swabs (97% vs. 54% dry-flocked swabs; 0.99 vs. 0.88 dry-flocked swabs, respectively). Sample collection using sponge-tipped swabs provides a DNA source of sufficient quantity and quality for pharmacogenetic variant detection using qPCR.

Manipulation of an existing crystal form unexpectedly results in interwoven packing networks with pseudo-translational symmetry.

Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes that synthesize a myriad of diverse molecules. Tailoring domains have been co-opted into NRPSs to introduce further variety into nonribosomal peptide products. Linear gramicidin synthetase contains a unique formylation-tailoring domain in its initiation module (F-A-PCP). The structure of the F-A di-domain has previously been determined in a crystal form which had large solvent channels and no density for the minor Asub subdomain. An attempt was made to take advantage of this packing by removing the Asub subdomain from the construct (F-AΔsub) in order to produce a crystal that could accommodate the PCP domain. In the resulting crystal the original packing network was still present, but a second network with the same packing and almost no contact with the original network took the place of the solvent channels and changed the space group of the crystal.

Introducing pharmacogenetic testing with clinical decision support into primary care: a feasibility study.

BACKGROUND: Inappropriate prescribing increases patient illness and death owing to adverse drug events. The inclusion of genetic information into primary care medication practices is one solution. Our aim was to assess the ability to obtain and genotype saliva samples and to determine the levels of use of a decision support tool that creates medication options adjusted for patient characteristics, drug-drug interactions and pharmacogenetics. METHODS: We conducted a cohort study in 6 primary care settings (5 family practices and 1 pharmacy), enrolling 191 adults with at least 1 of 10 common diseases. Saliva samples were obtained in the physician's office or pharmacy and sent to our laboratory, where DNA was extracted and genotyped and reports were generated. The reports were sent directly to the family physician/pharmacist and linked to an evidence-based prescribing decision support system. The primary outcome was ability to obtain and genotype samples. The secondary outcomes were yield and purity of DNA samples, ability to link results to decision support software and use of the decision support software. RESULTS: Genotyping resulted in linking of 189 patients (99%) with pharmacogenetic reports to the decision support program. A total of 96.8% of samples had at least 1 actionable genotype for medications included in the decision support system. The medication support system was used by the physicians and pharmacists 236 times over 3 months. INTERPRETATION: Physicians and pharmacists can collect saliva samples of sufficient quantity and quality for DNA extraction, purification and genotyping. A clinical decision support system with integrated data from pharmacogenetic tests may enable personalized prescribing within primary care. Trial registration: ClinicalTrials.gov, NCT02383290.