Enzyme engineering lets us play with new building blocks in non-ribosomal peptide synthesis.

In a recent issue of Nature Chemical Biology, Folger et al. demonstrated a high-throughput approach for engineering peptide bond forming domains from non-ribosomal peptide synthesis. A non-ribosomal peptide synthetase module from surfactin biosynthesis was reprogrammed to accept a fatty acid substrate into peptide biosynthesis, thus illustrating the potential of this approach for generating novel bioactive peptides.

An Engineered Biarylitide Cross-Linking P450 from RiPP Biosynthesis Generates Alternative Cyclic Peptides.

Cytochrome-P450-mediated cross-linking of ribosomally encoded peptides (RiPPs) is rapidly expanding and displays great potential for biocatalysis. Here, we demonstrate that active site engineering of the biarylitide cross-linking enzyme P450Blt enables the formation of His-X-Tyr and Tyr-X-Tyr cross-linked peptides, thus showing how such P450s can be further exploited to produce alternate cyclic tripeptides with controlled cross-linking states.

Resurrecting ancestral antibiotics: unveiling the origins of modern lipid II targeting glycopeptides.

Antibiotics are central to modern medicine, and yet they are mainly the products of intra and inter-kingdom evolutionary warfare. To understand how nature evolves antibiotics around a common mechanism of action, we investigated the origins of an extremely valuable class of compounds, lipid II targeting glycopeptide antibiotics (GPAs, exemplified by teicoplanin and vancomycin), which are used as last resort for the treatment of antibiotic resistant bacterial infections. Using a molecule-centred approach and computational techniques, we first predicted the nonribosomal peptide synthetase assembly line of paleomycin, the ancestral parent of lipid II targeting GPAs. Subsequently, we employed synthetic biology techniques to produce the predicted peptide and validated its antibiotic activity. We revealed the structure of paleomycin, which enabled us to address how nature morphs a peptide antibiotic scaffold through evolution. In doing so, we obtained temporal snapshots of key selection domains in nonribosomal peptide synthesis during the biosynthetic journey from ancestral, teicoplanin-like GPAs to modern GPAs such as vancomycin. Our study demonstrates the synergy of computational techniques and synthetic biology approaches enabling us to journey back in time, trace the temporal evolution of antibiotics, and revive these ancestral molecules. It also reveals the optimisation strategies nature has applied to evolve modern GPAs, laying the foundation for future efforts to engineer this important class of antimicrobial agents.

Beyond vancomycin: recent advances in the modification, reengineering, production and discovery of improved glycopeptide antibiotics to tackle multidrug-resistant bacteria.

Glycopeptide antibiotics (GPAs), which include vancomycin and teicoplanin, are important last-resort antibiotics used to treat multidrug-resistant Gram-positive bacterial infections. Whilst second-generation GPAs - generated through chemical modification of natural GPAs - have proven successful, the emergence of GPA resistance has underlined the need to develop new members of this compound class. Significant recent advances have been made in GPA research, including gaining an in-depth understanding of their biosynthesis, improving titre in production strains, developing new derivatives via novel chemical modifications and identifying a new mode of action for structurally diverse type-V GPAs. Taken together, these advances demonstrate significant untapped potential for the further development of GPAs to tackle the growing threat of multidrug-resistant bacteria.

Replacing Commercial 6-Phosphofructokinase in an Online Pyrophosphate Detection Assay.

Detection of pyrophosphate is important in quantifying enzyme activity, particularly adenylation domain activity during non-ribosomal peptide synthesis. The previous development of an enzyme coupled PPi /NADH assay allowed the measurement of such activity in an online fashion using commercially available components. Now, with a key enzyme - 6-phosphofructokinase - no longer available, we have screened and identified viable replacement enzymes that can be expressed in high yield and that are far superior in activity to the now discontinued commercial product. This will support the ability of groups to continue to use this established online assay for pyrophosphate detection.

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.

Smoking in pregnancy is associated with increased adiposity and retinal arteriolar wall-to-lumen ratio in adolescence: The Copenhagen Child Cohort Study 2000.

PURPOSE: To investigate the association between prenatal exposures and anthropometric data and cardiovascular risk factors including retinal arteriolar wall-to-lumen ratio in adolescence. METHODS: This longitudinal observational study included all 1445 adolescents from the Copenhagen Child Cohort 2000 who attended the 2016-2017 examination. Outcome measures included retinal arteriolar wall-to-lumen ratio, height, body mass index, waist-to-hip ratio, body composition measured by bioimpedance, and blood pressure. Information on prenatal exposures (birth weight, gestational age, maternal smoking during pregnancy) as well as sex, parental age, household income and parental educational levels were obtained from national registries. Associations between exposures and outcome measures were analyzed using general linear models. RESULTS: Maternal smoking during pregnancy was associated with a higher retinal arteriolar wall-to-lumen ratio (0.004 or 1.9%, P = 0.009) at age 16/17 years, an association driven exclusively by the female participants (0.008 or 3.7%, P < 0.0001). Maternal smoking during pregnancy was also associated to higher body-mass index (1.43 kg/m2, P < 0.0001), waist-to-hip ratio (0.02, P < 0.0001) and fat mass index (0.93 kg/m2, P < 0.0001). Birth weight, gestational age, and parental age had no detectable impact on retinal arteriolar wall-to-lumen ratios. CONCLUSION: Prenatal exposure to tobacco smoking is associated with a higher risk of obesity and, predominantly in girls, to a greater retinal arteriolar wall thickness, which suggests that maternal smoking may induce an unfavorable cardiovascular and metabolic risk profile in the child.

Progression Over 5 Years of Prelaminar Hyperreflective Lines to Optic Disc Drusen in the Copenhagen Child Cohort 2000 Eye Study.

BACKGROUND: The purpose of the study was to examine 5-year changes in eyes with optic disc drusen at baseline on optical coherence tomography (OCT) scans and the relation of incident drusen to hyperreflective prelaminar lines. METHODS: The study included children who presented at baseline, when participants were aged 11-12 years, and again 5 years later. Grading for optic disc drusen was made in all. Grading for prelaminar lines was made in all children at follow-up and in eyes with optic disc drusen at baseline. Analyses included associations with scleral canal diameter at baseline in all children with optic disc drusen and a nested control group of 115 children without optic disc drusen. Data are reported as the number of children having at least one drusen or at least one hyperreflective line per person. RESULTS: The analysis included 724 children who attended both rounds of the study. Of these, 11 (1.5%) had optic disc drusen at baseline. Five additional children had developed optic disc drusen at follow-up, whereas optic disc drusen had disappeared in none, so that 16 (2.2%) children had optic disc drusen in one or both eyes at follow-up. Children with optic disc drusen at the 5-year follow-up had had a mean scleral canal diameter of 1,364 µm (interquartile range [IQR] 81 µm), compared with 1,457 µm (IQR 197) µm in 115 nested controls without optic disc drusen (P < 0.001). Optic disc drusen at follow-up were associated with more hypermetropic refraction. All children who had optic disc drusen at follow-up also had prelaminar hyperreflective lines. In addition, such lines were found at follow-up in 24 of the remaining 708 children without optic disc drusen (P < 0.001). Prelaminar hyperreflective lines with or without optic disc drusen were associated with a narrower scleral canal (diameter 1,364 µm, IQR 119 µm) compared with absence of prelaminar lines (1,486 µm, IQR 206 µm; P < 0.0001). CONCLUSION: This study provides the first evidence from a prospective study that small optic discs and prelaminar hyperreflective lines on OCT are risk factors for the development of optic disc drusen. The association between prelaminar hyperreflective lines, hypermetropia, and a narrow scleral canal supports that a crowded disc is an essential predisposing factor for the development of optic disc drusen.

Retinal arteriolar wall-to-lumen ratios at 16-17 years in the Copenhagen Child Cohort 2000 Study.

PURPOSE: To study the thickness of retinal arteriolar walls in a population-based cohort of adolescents. METHODS: This cross-sectional, observational study included 1217 participants aged 16-17 years from the Copenhagen Child Cohort 2000 Study. The wall thickness and lumen diameter of a major branch retinal arteriole were measured using adaptive optics imaging. The wall-to-lumen ratio was analyzed in relation to blood pressure and body composition variables using a general linear model. Overall in the study population, wall-to-lumen ratio was found to decrease by 0.49% per µm increase in arteriole diameter (P < 0.0001) and all subsequent analyzes were adjusted accordingly. RESULTS: The average outer and inner arteriole diameters were 117 ±â€Š19 and 96.6 ±â€Š18 µm (mean ±â€ŠSD), corresponding to a wall-to-lumen ratio of 0.21 ±â€Š0.024. There was no detectable difference between sexes. A higher wall-to-lumen ratio was associated with a higher BMI (+0.21% per kg/m, P = 0.0018), higher body fat percentage (+0.097% per 1% increase, P = 0.0052), wider hip circumference (+1.1% per 10 cm increase, P = 0.0006), wider waist circumference (+0.92% per 10 cm increase, P = 0.0009), higher SBP in girls (+1.1% per 10 mmHg increase, P = 0.0005), longer axial length (+0.70% per mm increase, P = 0.013), and younger age (+4.9% per year younger, P < 0.0001), adjusted for arteriole diameter, age, sex, and height. CONCLUSION: A higher retinal arteriolar wall-to-lumen ratio was associated with all registered indices of body fat proportion.