Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity.

Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5-9.0 and temperatures from 30-70 °C. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.

Agreement between self-reported asthma symptoms and exhaled nitric oxide levels: impact on inhaled corticosteroid prescribing in general practice. An observational study.

BACKGROUND: The National Review of Asthma Deaths UK highlighted that 46% of deaths could be avoided and recommended that all sufferers receive a structured asthma annual review which assess asthma control. In primary care this is commonly achieved using symptom-based questionnaires such as the Asthma Control Test (ACT). A newer method of assessing asthma control is Fractional Exhaled Nitric Oxide (FeNO) testing, which is currently recommended for the diagnosis of asthma, but not for monitoring of asthma control. The study aim was to assess the correlation between self-reported symptoms as measured by the ACT and FeNO testing and the subsequent impact of FeNO testing on prescribing of asthma medication. METHODS: A retrospective review of 65 patients who had received both ACT and FeNO testing as part of their asthma annual review. A spearman correlation was used to estimate the correlation between ACT scores and FENO levels. A χ2 test was used to compare prompting frequency of the measures and Kendalls τ statistic was made to estimate their concordance and influence on subsequent ICS medication prescription. RESULTS: The mean age of the participants was 41 years (4-93 years). There was no statistically significant correlation between ACT and FeNO (ρ = 0.195, p = 0.120). The median FeNO was 26 ppb (range 8-279 ppb), and the ACT score 20 (range 5 to 25 points). Furthermore, FeNO more frequently prompts a change in medication than ACT, 66% versus 42% (p = 0.005). A low concordance between the measures was found (Kendall's τ statistic - 0.321). CONCLUSION: FeNO should be considered for monitoring of control in asthma. To balance the cost of implementing this technology into primary care a risk stratified approach could be applied to testing.

Co-crystallization of human inositol monophosphatase with the lithium mimetic L-690,330.

Lithium, which is still the gold standard in the treatment of bipolar disorder, has been proposed to inhibit inositol monophosphatase (IMPase) and is hypothesized to exert its therapeutic effects by attenuating phosphatidylinositol (PI) cell signalling. Drug-discovery efforts have focused on small-molecule lithium mimetics that would specifically inhibit IMPase without exhibiting the undesired side effects of lithium. L-690,330 is a potent bisphosphonate substrate-based inhibitor developed by Merck Sharp & Dohme. To aid future structure-based inhibitor design, determination of the exact binding mechanism of L-690,330 to IMPase was of interest. Here, the high-resolution X-ray structure of human IMPase in complex with L690,330 and manganese ions determined at 1.39 Šresolution is reported.

Definitive Zygosity Scores in the Peas in the Pod Questionnaire is a Sensitive and Accurate Assessment of the Zygosity of Adult Twins.

Twin researchers face the challenge of accurately determining the zygosity of twins for research. As part of the annual questionnaire between 1999 and 2006, 8,307 twins from the TwinsUK registry were asked to complete five questions (independently from their co-twin) to ascertain their self-perceived zygosity during childhood on up to five separate occasions. This questionnaire is known as the 'peas in the pod' questionnaire (PPQ), but there is little evidence of its validation. Answers were scored and classified as monozygotic (MZ), dizygotic (DZ), or unknown zygosity (UZ) and were compared with 4,484 twins with genotyping data who had not been selected for zygosity. Of these, 3,859 individuals (46.5% of those who had a zygosity from PPQ) had zygosity classified by both the PPQ and genotyping. Of the 708 individual twins whose answers meant that they were consistently classed as MZ in the PPQ, 683 (96.5%) were MZ within the genotype data. Of the 945 individual twins consistently classed as DZ within questionnaire, 936 (99.0%) were DZ in the genotype data. Where both twins scored MZ consistently across multiple questionnaires, 99.6% were MZ on genotyping, 99.7% were DZ on genotyping if both twins consistently scored DZ. However, for the initial questionnaire, 88.6% of those scoring as MZ were genotypically MZ and 98.7% DZ. For twin pairs where both scored UZ, 94.7% were DZ. Using the PPQ on a single occasion provided a definitive classification of whether the twin was MZ or DZ with an overall accuracy of 86.9%, increasing to 97.9% when there was a consistent classification of zygosity across multiple questionnaires. This study has shown that the PPQ questionnaire is an excellent proxy indicator of zygosity in the absence of genotyping information.

The rate of nonallelic homologous recombination in males is highly variable, correlated between monozygotic twins and independent of age.

Nonallelic homologous recombination (NAHR) between highly similar duplicated sequences generates chromosomal deletions, duplications and inversions, which can cause diverse genetic disorders. Little is known about interindividual variation in NAHR rates and the factors that influence this. We estimated the rate of deletion at the CMT1A-REP NAHR hotspot in sperm DNA from 34 male donors, including 16 monozygotic (MZ) co-twins (8 twin pairs) aged 24 to 67 years old. The average NAHR rate was 3.5 × 10(-5) with a seven-fold variation across individuals. Despite good statistical power to detect even a subtle correlation, we observed no relationship between age of unrelated individuals and the rate of NAHR in their sperm, likely reflecting the meiotic-specific origin of these events. We then estimated the heritability of deletion rate by calculating the intraclass correlation (ICC) within MZ co-twins, revealing a significant correlation between MZ co-twins (ICC = 0.784, p = 0.0039), with MZ co-twins being significantly more correlated than unrelated pairs. We showed that this heritability cannot be explained by variation in PRDM9, a known regulator of NAHR, or variation within the NAHR hotspot itself. We also did not detect any correlation between Body Mass Index (BMI), smoking status or alcohol intake and rate of NAHR. Our results suggest that other, as yet unidentified, genetic or environmental factors play a significant role in the regulation of NAHR and are responsible for the extensive variation in the population for the probability of fathering a child with a genomic disorder resulting from a pathogenic deletion.

Unravelling the basis of variability in cobalamin levels in the general population.

Cobalamin (Cbl) is an essential B vitamin involved in the normal functioning of the nervous system, the formation of key components of blood, DNA synthesis and methylation, and energy production. Physiological levels of Cbl vary greatly within populations, although the basis for this variability remains largely unknown. We conducted a twin study to characterise the basis of variation in plasma Cbl levels and to test whether common genetic polymorphisms in genes known to cause defects in inborn errors of Cbl metabolism and transport are also associated with mean plasma Cbl levels in the general population. The present results showed that plasma levels of Cbl were heritable, with genetic and phenotypic variance increasing with age, and levels significantly correlated with age, BMI, exercise, alcohol consumption, smoking status, social class and folate levels, which collectively accounted for up to 15 % of Cbl variation. Of eight genes responsible for the defects of the Cbl metabolic pathway (cblA-G and mut), MMAA, MMACHC, MTRR and MUT harboured polymorphisms that showed evidence of association with Cbl levels. Characterisation of the heritable component of variation in Cbl levels can facilitate the early diagnosis and prognosis of Cbl insufficiency/deficiency in individuals at a higher risk of associated diseases.

Genome-wide association study reveals a complex genetic architecture underpinning-induced CYP3A4 enzyme activity.

Atypical cytochrome P450 3A4 (CYP3A4) enzyme activity-induced and inhibited-is thought to be the driver of numerous poor or adverse therapeutic responses to up to 50 % of all commonly prescribed drugs. We carried out a genome-wide association study to identify common genetic variants associated with variation in induced CYP3A4 activity. A total of 310 twins were included in this study. Each participant had already completed a 14 days course of St John's Wort to induce CYP3A4, which was quantified through the metabolic ratio of exogenous 3-hydroxyquinine to quinine. We failed to detect any genome-wide significant associations (P < 1 × 10(-8)) with variation in induced CYP3A4 activity although several genomic regions were highlighted which may play minor roles. We report the first GWAS of variation in induced CYP3A4 activity and our preliminary results indicate a complex genetic architecture underpinning induced CYP3A4 enzyme activity.

A twin study of mitochondrial DNA polymorphisms shows that heteroplasmy at multiple sites is associated with mtDNA variant 16093 but not with zygosity.

The mitochondrial theory of ageing proposes that damage to mitochondria and diminished mitochondrial DNA (mtDNA) repair are major contributors to cellular dysfunction and age-related diseases. We investigate the prevalence of heteroplasmy in the mtDNA control region in buccal swab and blood derived samples for 178 women from the TwinsUK cohort (41 DZ pair 39 MZ pairs, 18 singletons, mean age 57.5 range 28-82) and its relationship to age, BMI and fasting insulin and glucose serum levels. The overall estimated prevalence of heteroplasmy for both tissues in the control region measured for 37 sites was 17%. The prevalence of heteroplasmy was higher among the older half of the study subjects than in the younger half (23% vs 10% p<0.03), primarily reflecting the increase in the prevalence of a heteroplasmic dinucleotide CA repeat in variable region II (VRII) with age. The VRII 523-524 heteroplasmic site (heteroplasmic in 25 subjects) was also associated with a decrease in BMI. In addition, concordance rates for common heteroplasmy were observed to be near complete for both dizygotic (DZ = 94%) and monozygotic twin pairs (MZ = 100%), consistent with previous reports that suggest variation in heteroplasmy rates between generations are determined by bottlenecks in maternal transmission of mitochondria. Differences in the prevalence of heteroplasmy were observed overall between samples derived from buccal swabs (19%) and blood (15%, p<0.04). These were particularly marked at position 16093 of hypervariable region I (HVI, 7% vs 0%, respectively, p<4×10(-11)). The presence of the C allele at position 16093 in blood was associated with the presence of heteroplasmy in buccal swabs at this position (p = 3.5×10(-14)) and also at VRII (p = 2×10(-4)) suggesting a possible predisposing role for this site in the accumulation of heteroplasmy. Our data indicate that BMI is potentially associated with control region heteroplasmy.

Genetic epidemiology of induced CYP3A4 activity.

AIM: The cytochrome P450 3A4 (CYP3A4) enzyme is implicated in the metabolism of more than 50% of all prescribed medications and its activity - including induced or inhibited activity - is deemed to be a crucial determinant of interindividual variability in drug disposition, poor therapeutic efficacy, and adverse response to medication. METHODS: We used the classical twin model in conjunction with an induction experiment to uncover the relative contribution of genetic and environmental factors to interindividual variation in induced CYP3A4 activity. A total of 367 healthy twins participated in the study. Each volunteer was administered a potent inducer of CYP3A4 (St John's Wort) for 14 days and the activity of CYP3A4 was quantified through the metabolism of the exogenously administered probe drug quinine sulfate. RESULTS: Baseline and induced CYP3A4 activity were highly variable with a seven-fold and 11-fold difference among our population, respectively. Alcohol consumption, BMI, and smoking were significantly associated with induced CYP3A4 activity, collectively explaining 20% of the variation (P<1×10(-4)). The narrow-sense heritability of induced CYP3A4 activity was estimated at 66%, whereas the remainder of the variation was attributed to unique environmental factors. CONCLUSION: To our knowledge, this is the first genetic epidemiological study of induced CYP3A4 activity. Our results motivate further research to identify common and rarer genetic variants that underpin the heritable component of variation in induced CYP3A4 activity.

The basis of differential responses to folic acid supplementation.

BACKGROUND/AIMS: Elevated levels of total homocysteine (tHcy) are associated with an increased risk of many common diseases. Supplementation with folic acid has been shown to significantly reduce tHcy levels. We used the classical twin model to partition the variability in changes in plasma tHcy levels through folic acid supplementation into genetic, environmental, and confounding epidemiological factors. METHODS: We carried out an intervention study of folic acid using 101 healthy, female, identical and non-identical twins aged 50-80 years. Each twin was administered folic acid (0.8 mg/day) for 6 weeks. Total plasma folate, cobalamin and tHcy were measured at both baseline and after dosing. We calculated the heritability and tested for associations between the MTHFR C677T functional variant and response to folic acid supplementation. RESULTS: Supplementation with folic acid led to a significant reduction in tHcy levels. The mean tHcy changed from 12.14 to 10.42 µmol/l after supplementation (p < 10(-5)). Moreover, the change in tHcy levels was highly heritable (64%), not associated with the C677T functional variant at MTHFR and not confounded by age, BMI or diet. CONCLUSIONS: Our results highlight the need to identify genetic factors associated with biomarkers of response to folate supplementation.

A structural study of norovirus 3C protease specificity: binding of a designed active site-directed peptide inhibitor.

Noroviruses are the major cause of human epidemic nonbacterial gastroenteritis. Viral replication requires a 3C cysteine protease that cleaves a 200 kDa viral polyprotein into its constituent functional proteins. Here we describe the X-ray structure of the Southampton norovirus 3C protease (SV3CP) bound to an active site-directed peptide inhibitor (MAPI) which has been refined at 1.7 Å resolution. The inhibitor, acetyl-Glu-Phe-Gln-Leu-Gln-X, which is based on the most rapidly cleaved recognition sequence in the 200 kDa polyprotein substrate, reacts covalently through its propenyl ethyl ester group (X) with the active site nucleophile, Cys 139. The structure permits, for the first time, the identification of substrate recognition and binding groups in a noroviral 3C protease and thus provides important new information for the development of antiviral prophylactics.

Trapping of palindromic ligands within native transthyretin prevents amyloid formation.

Transthyretin (TTR) amyloidosis is a fatal disease for which new therapeutic approaches are urgently needed. We have designed two palindromic ligands, 2,2'-(4,4'-(heptane-1,7-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (mds84) and 2,2'-(4,4'-(undecane-1,11-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (4ajm15), that are rapidly bound by native wild-type TTR in whole serum and even more avidly by amyloidogenic TTR variants. One to one stoichiometry, demonstrable in solution and by MS, was confirmed by X-ray crystallographic analysis showing simultaneous occupation of both T4 binding sites in each tetrameric TTR molecule by the pair of ligand head groups. Ligand binding by native TTR was irreversible under physiological conditions, and it stabilized the tetrameric assembly and inhibited amyloidogenic aggregation more potently than other known ligands. These superstabilizers are orally bioavailable and exhibit low inhibitory activity against cyclooxygenase (COX). They offer a promising platform for development of drugs to treat and prevent TTR amyloidosis.

Structure of human porphobilinogen deaminase at 2.8 A: the molecular basis of acute intermittent porphyria.

Mutations in the human PBGD (porphobilinogen deaminase) gene cause the inherited defect AIP (acute intermittent porphyria). In the present study we report the structure of the human uPBGD (ubiquitous PBGD) mutant, R167Q, that has been determined by X-ray crystallography and refined to 2.8 A (1 A=0.1 nm) resolution (Rfactor=0.26, Rfree=0.29). The protein crystallized in space group P2(1)2(1)2 with two molecules in the asymmetric unit (a=81.0 A, b=104.4 A and c=109.7 A). Phases were obtained by molecular replacement using the Escherichia coli PBGD structure as a search model. The human enzyme is composed of three domains each of approx. 110 amino acids and possesses a dipyrromethane cofactor at the active site, which is located between domains 1 and 2. An ordered sulfate ion is hydrogen-bonded to Arg26 and Ser28 at the proposed substrate-binding site in domain 1. An insert of 29 amino acid residues, present only in mammalian PBGD enzymes, has been modelled into domain 3 where it extends helix alpha2(3) and forms a beta-hairpin structure that contributes to a continuous hydrogen-bonding network spanning domains 1 and 3. The structural and functional implications of the R167Q mutation and other mutations that result in AIP are discussed.

X-ray, neutron and NMR studies of the catalytic mechanism of aspartic proteinases.

Current proposals for the catalytic mechanism of aspartic proteinases are largely based on X-ray structures of bound oligopeptide inhibitors possessing non-hydrolysable analogues of the scissile peptide bond. Until recent years, the positions of protons on the catalytic aspartates and the ligand in these complexes had not been determined with certainty due to the inadequate resolution of these analyses. There has been much interest in locating the catalytic protons at the active site of aspartic proteinases since this has major implications for detailed understanding of the mechanism of action and the design of improved transition state mimics for therapeutic applications. In this review we discuss the results of studies which have shed light on the locations of protons at the catalytic centre. The first direct determination of the proton positions stemmed from neutron diffraction data collected from crystals of the fungal aspartic proteinase endothiapepsin bound to a transition state analogue (H261). The neutron structure of the complex at a resolution of 2.1 A provided evidence that Asp 215 is protonated and that Asp 32 is the negatively charged residue in the transition state complex. Atomic resolution X-ray studies of inhibitor complexes have corroborated this finding. A similar study of the native enzyme established that it, unexpectedly, has a dipeptide bound at the catalytic site which is consistent with classical reports of inhibition by short peptides and the ability of pepsins to catalyse transpeptidation reactions. Studies by NMR have confirmed the findings of low-barrier and single-well hydrogen bonds in the complexes with transition state analogues.

The 1.6A X-ray structure of the unusual c-type cytochrome, cytochrome cL, from the methylotrophic bacterium Methylobacterium extorquens.

The structure of cytochrome cL from Methylobacterium extorquens has been determined by X-ray crystallography to a resolution of 1.6 A. This unusually large, acidic cytochrome is the physiological electron acceptor for the quinoprotein methanol dehydrogenase in the periplasm of methylotrophic bacteria. Its amino acid sequence is completely different from that of other cytochromes but its X-ray structure reveals a core that is typical of class I cytochromes c, having alpha-helices folded into a compact structure enclosing the single haem c prosthetic group and leaving one edge of the haem exposed. The haem is bound through thioether bonds to Cys65 and Cys68, and the fifth ligand to the haem iron is provided by His69. Remarkably, the sixth ligand is provided by His112, and not by Met109, which had been shown to be the sixth ligand in solution. Cytochrome cL is unusual in having a disulphide bridge that tethers the long C-terminal extension to the body of the structure. The crystal structure reveals that, close to the inner haem propionate, there is tightly bound calcium ion that is likely to be involved in stabilization of the redox potential, and that may be important in the flow of electrons from reduced pyrroloquinoline quinone in methanol dehydrogenase to the haem of cytochrome cL. As predicted, both haem propionates are exposed to solvent, accounting for the unusual influence of pH on the redox potential of this cytochrome.

High-resolution structure of myo-inositol monophosphatase, the putative target of lithium therapy.

Inositol monophosphatase is a key enzyme of the phosphatidylinositol signalling pathway and the putative target of the mood-stabilizing drug lithium. The crystal structure of bovine inositol monophosphatase has been determined at 1.4 A resolution in complex with the physiological magnesium ion ligands. Three magnesium ions are octahedrally coordinated at the active site of each of the two subunits of the inositol monophosphatase dimer and a detailed three-metal mechanism is proposed. Ligands to the three metals include the side chains of Glu70, Asp90, Asp93 and Asp220, the backbone carbonyl group of Ile92 and several solvent molecules, including the proposed nucleophilic water molecule (W1) ligated by both Mg-1 and Mg-3. Modelling of the phosphate moiety of inositol monophosphate to superpose the axial phosphate O atoms onto three active-site water molecules orientates the phosphoester bond for in-line attack by the nucleophilic water which is activated by Thr95. Modelling of the pentacoordinate transition state suggests that the 6-OH group of the inositol moiety stabilizes the developing negative charge by hydrogen bonding to a phosphate O atom. Modelling of the post-reaction complex suggests a role for a second water molecule (W2) ligated by Mg-2 and Asp220 in protonating the departing inositolate. This second water molecule is absent in related structures in which lithium is bound at site 2, providing a rationale for enzyme inhibition by this simple monovalent cation. The higher resolution structural information on the active site of inositol monophosphatase will facilitate the design of substrate-based inhibitors and aid in the development of better therapeutic agents for bipolar disorder (manic depression).

The X-ray structure of the plant like 5-aminolaevulinic acid dehydratase from Chlorobium vibrioforme complexed with the inhibitor laevulinic acid at 2.6 A resolution.

5-Aminolaevulinic acid dehydratase (ALAD), an early enzyme of the tetrapyrrole biosynthesis pathway, catalyses the dimerisation of 5-aminolaevulinic acid to form the pyrrole, porphobilinogen. ALAD from Chlorobium vibrioforme is shown to form a homo-octameric structure with 422 symmetry in which each subunit adopts a TIM-barrel fold with a 30 residue N-terminal arm extension. Pairs of monomers associate with their arms wrapped around each other. Four of these dimers interact principally via their arm regions to form octamers in which each active site is located on the surface. The active site contains two invariant lysine residues (200 and 253), one of which (Lys253) forms a Schiff base link with the bound substrate analogue, laevulinic acid. The carboxyl group of the laevulinic acid forms hydrogen bonds with the side-chains of Ser279 and Tyr318. The structure was examined to determine the location of the putative active-site magnesium ion, however, no evidence for the metal ion was found in the electron density map. This is in agreement with previous kinetic studies that have shown that magnesium stimulates but is not required for activity. A different site close to the active site flap, in which a putative magnesium ion is coordinated by a glutamate carboxyl and five solvent molecules may account for the stimulatory properties of magnesium ions on the enzyme.

Atomic resolution analysis of the catalytic site of an aspartic proteinase and an unexpected mode of binding by short peptides.

The X-ray structures of native endothiapepsin and a complex with a hydroxyethylene transition state analog inhibitor (H261) have been determined at atomic resolution. Unrestrained refinement of the carboxyl groups of the enzyme by using the atomic resolution data indicates that both catalytic aspartates in the native enzyme share a single negative charge equally; that is, in the crystal, one half of the active sites have Asp 32 ionized and the other half have Asp 215 ionized. The electron density map of the native enzyme refined at 0.9 A resolution demonstrates that there is a short peptide (probably Ser-Thr) bound noncovalently in the active site cleft. The N-terminal nitrogen of the dipeptide interacts with the aspartate diad of the enzyme by hydrogen bonds involving the carboxyl of Asp 215 and the catalytic water molecule. This is consistent with classical findings that the aspartic proteinases can be inhibited weakly by short peptides and that these enzymes can catalyze transpeptidation reactions. The dipeptide may originate from autolysis of the N-terminal Ser-Thr sequence of the enzyme during crystallization.

Flux assays in high throughput screening of ion channels in drug discovery.

Ion channels have been identified as therapeutic targets in various disorders, such as cardiovascular disease, neurological disease, and cystic fibrosis. Flux assays to detect functional ionic flux through ion channels are becoming increasingly popular as tools for screening compounds. In an optimized flux assay, modulation of ion channel activity may produce readily detectable changes in radiolabeled or nonradiolabeled ionic flux. Technologies based on flux assays are currently available in a fully automated high throughput format for efficient screening. This application offers sensitive, precise, and reproducible measurements giving accurate drug rank orders matching those of patch clamp data. Conveniently, the flux assay is amenable to adaptation for different ion channels, such as potassium, sodium, calcium, and chloride channels, by using suitable tracer ions. The nonradiolabeled rubidium-based flux assay coupled with the ion channel reader (ICR) technology has become very successful in ion channel activity analysis and is emerging as a popular technique in modern drug discovery.