Cytochrome P450 (CYP) enzymes play key roles in drug metabolism and adverse drug-drug interactions. Despite tremendous efforts in the past decades, essential questions regarding the function and activity of CYPs remain unanswered. Here, we used a combination of sequence-based co-evolutionary analysis and structure-based anisotropic thermal diffusion (ATD) molecular dynamics simulations to detect allosteric networks of amino acid residues and characterize their biological and molecular functions. We investigated four CYP subfamilies (CYP1A, CYP2D, CYP2C, and CYP3A) that are involved in 90% of all metabolic drug transformations and identified four amino acid interaction networks associated with specific CYP functionalities, i.e., membrane binding, heme binding, catalytic activity, and dimerization. Interestingly, we did not detect any co-evolved substrate-binding network, suggesting that substrate recognition is specific for each subfamily. Analysis of the membrane binding networks revealed that different CYP proteins adopt different membrane-bound orientations, consistent with the differing substrate preference for each isoform. The catalytic networks were associated with conservation of catalytic function among CYP isoforms, whereas the dimerization network was specific to different CYP isoforms. We further applied low-temperature ATD simulations to verify proposed allosteric sites associated with the heme-binding network and their role in regulating metabolic fate. Our approach allowed for a broad characterization of CYP properties, such as membrane interactions, catalytic mechanisms, dimerization, and linking these to groups of residues that can serve as allosteric regulators. The presented combined co-evolutionary analysis and ATD simulation approach is also generally applicable to other biological systems where allostery plays a role.
The α-proteobacterium Wolbachia pipientis infects more than 65% of insect species worldwide and manipulates the host reproductive machinery to enable its own survival. It can live in mutualistic relationships with hosts that cause human disease, including mosquitoes that carry the Dengue virus. Like many other bacteria, Wolbachia contains disulfide bond forming (Dsb) proteins that introduce disulfide bonds into secreted effector proteins. The genome of the Wolbachia strain wMel encodes two DsbA-like proteins sharing just 21% sequence identity to each other, α-DsbA1 and α-DsbA2, and an integral membrane protein, α-DsbB. α-DsbA1 and α-DsbA2 both have a Cys-X-X-Cys active site that, by analogy with Escherichia coli DsbA, would need to be oxidized to the disulfide form to serve as a disulfide bond donor toward substrate proteins. Here we show that the integral membrane protein α-DsbB oxidizes α-DsbA1, but not α-DsbA2. The interaction between α-DsbA1 and α-DsbB is very specific, involving four essential cysteines located in the two periplasmic loops of α-DsbB. In the electron flow cascade, oxidation of α-DsbA1 by α-DsbB is initiated by an oxidizing quinone cofactor that interacts with the cysteine pair in the first periplasmic loop. Oxidizing power is transferred to the second cysteine pair, which directly interacts with α-DsbA1. This reaction is inhibited by a non-catalytic disulfide present in α-DsbA1, conserved in other α-proteobacterial DsbAs but not in γ-proteobacterial DsbAs. This is the first characterization of the integral membrane protein α-DsbB from Wolbachia and reveals that the non-catalytic cysteines of α-DsbA1 regulate the redox relay system in cooperation with α-DsbB.
The study describes a comparative analysis of biochemical, structural and functional properties of two recombinant derivatives from Clostridium thermocellum ATCC 27405 belonging to family 43 glycoside hydrolase. The family 43 glycoside hydrolase encoding α-L-arabinofuranosidase (Ct43Araf) displayed an N-terminal catalytic module CtGH43 (903 bp) followed by two carbohydrate binding modules CtCBM6A (405 bp) and CtCBM6B (402 bp) towards the C-terminal. Ct43Araf and its truncated derivative CtGH43 were cloned in pET-vectors, expressed in Escherichia coli and functionally characterized. The recombinant proteins displayed molecular sizes of 63 kDa (Ct43Araf) and 34 kDa (CtGH43) on SDS-PAGE analysis. Ct43Araf and CtGH43 showed optimal enzyme activities at pH 5.7 and 5.4 and the optimal temperature for both was 50°C. Ct43Araf and CtGH43 showed maximum activity with rye arabinoxylan 4.7 Umg−1 and 5.0 Umg−1, respectively, which increased by more than 2-fold in presence of Ca2+ and Mg2+ salts. This indicated that the presence of CBMs (CtCBM6A and CtCBM6B) did not have any effect on the enzyme activity. The thin layer chromatography and high pressure anion exchange chromatography analysis of Ct43Araf hydrolysed arabinoxylans (rye and wheat) and oat spelt xylan confirmed the release of L-arabinose. This is the first report of α-L-arabinofuranosidase from C. thermocellum having the capacity to degrade both p-nitrophenol-α-L-arabinofuranoside and p-nitrophenol-α-L-arabinopyranoside. The protein melting curves of Ct43Araf and CtGH43 demonstrated that CtGH43 and CBMs melt independently. The presence of Ca2+ ions imparted thermal stability to both the enzymes. The circular dichroism analysis of CtGH43 showed 48% β-sheets, 49% random coils but only 3% α-helices.
Sulfation patterns along glycosaminoglycan (GAG) chains dictate their functional role. The N-deacetylase N-sulfotransferase family (NDST) catalyzes the initial downstream modification of heparan sulfate and heparin chains by removing acetyl groups from subsets of N-acetylglucosamine units and, subsequently, sulfating the residual free amino groups. These enzymes transfer the sulfuryl group from 3′-phosphoadenosine-5′-phosphosulfate (PAPS), yielding sulfated sugar chains and 3′-phosphoadenosine-5′-phosphate (PAP). For the N-sulfotransferase domain of NDST1, Lys833 has been implicated to play a role in holding the substrate glycan moiety close to the PAPS cofactor. Additionally, Lys833 together with His716 interact with the sulfonate group, stabilizing the transition state. Such a role seems to be shared by Lys614 through donation of a proton to the bridging oxygen of the cofactor, thereby acting as a catalytic acid. However, the relevance of these boundary residues at the hydrophobic cleft is still unclear. Moreover, whether Lys833, His716 and Lys614 play a role in both glycan recognition and glycan sulfation remains elusive. In this study we evaluate the contribution of NDST mutants (Lys833, His716 and Lys614) to dynamical effects during sulfate transfer using comprehensive combined docking and essential dynamics. In addition, the binding location of the glycan moiety, PAPS and PAP within the active site of NDST1 throughout the sulfate transfer were determined by intermediate state analysis. Furthermore, NDST1 mutants unveiled Lys833 as vital for both the glycan binding and subsequent N-sulfotransferase activity of NDST1.
The indole-diterpene paxilline is an abundant secondary metabolite synthesized by Penicillium paxilli. In total, 21 genes have been identified at the PAX locus of which six have been previously confirmed to have a functional role in paxilline biosynthesis. A combination of bioinformatics, gene expression and targeted gene replacement analyses were used to define the boundaries of the PAX gene cluster. Targeted gene replacement identified seven genes, paxG, paxA, paxM, paxB, paxC, paxP and paxQ that were all required for paxilline production, with one additional gene, paxD, required for regular prenylation of the indole ring post paxilline synthesis. The two putative transcription factors, PP104 and PP105, were not co-regulated with the pax genes and based on targeted gene replacement, including the double knockout, did not have a role in paxilline production. The relationship of indole dimethylallyl transferases involved in prenylation of indole-diterpenes such as paxilline or lolitrem B, can be found as two disparate clades, not supported by prenylation type (e.g., regular or reverse). This paper provides insight into the P. paxilli indole-diterpene locus and reviews the recent advances identified in paxilline biosynthesis.
indole-diterpene; paxilline; prenylation
Obesity may cluster in families due to shared physical and social environments.
This study aims to identify family typologies of obesity risk based on family environments.
Using 2007–2008 data from 706 parent/youth dyads in Minnesota, we applied latent profile analysis and general linear models to evaluate associations between family typologies and body mass index (BMI) of youth and parents.
Three typologies described most families with 18.8% “Unenriched/Obesogenic,” 16.9% “Risky Consumer,” and 64.3% “Healthy Consumer/Salutogenic.” After adjustment for demographic and socioeconomic factors, parent BMI and youth BMI Z-scores were higher in unenriched/obesogenic families (BMI difference=2.7, p<0.01 and BMI Z-score difference=0.51, p<0.01, respectively) relative to the healthy consumer/salutogenic typology. In contrast, parent BMI and youth BMI Z-scores were similar in the risky consumer families relative to those in healthy consumer/salutogenic type.
We can identify family types differing in obesity risks with implications for public health interventions.
Latent profile analysis; Family types; Youth; Obesogenic environment
Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C), and then to proline via pyrroline-5-carboxylate reductases (PYCRs). Here, the role of three isozymic versions of PYCR was addressed in human melanoma cells by tracking the fate of 13C-labeled precursors. Based on these studies we conclude that PYCR1 and PYCR2, which are localized in the mitochondria, are primarily involved in conversion of glutamate to proline. PYCRL, localized in the cytosol, is exclusively linked to the conversion of ornithine to proline. This analysis provides the first clarification of the role of PYCRs to proline biosynthesis.
Pyridoxal 5′-phosphate (PLP) is a cofactor for dozens of B6 requiring enzymes. PLP reacts with apo-B6 enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B6 enzyme. During protein turnover, the PLP is salvaged by first converting it to pyridoxal by a phosphatase and then back to PLP by pyridoxal kinase. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 4′-aldehyde moiety forms covalent adducts with other compounds and non-B6 proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complex with the product PLP to form an inactive enzyme complex. Evidence is presented that, in the inhibited complex, PLP has formed an aldimine bond with an active site lysine residue during catalytic turnover. The rate of dissociation of PLP from the complex is very slow, being only partially released after a 2-hour incubation with PLP phosphatase. Interestingly, the inactive pyridoxal kinase•PLP complex can be partially reactivated by transferring the tightly bound PLP to an apo-B6 enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell.
Proteases play an essential part in a variety of biological processes. Besides their importance under healthy conditions they are also known to have a crucial role in complex diseases like cancer. In recent years, it has been shown that not only the fragments produced by proteases but also their dynamics, especially ex vivo, can serve as biomarkers. But so far, only a few approaches were taken to explicitly model the dynamics of proteolysis in the context of mass spectrometry.
We introduce a new concept to model proteolytic processes, the degradation graph. The degradation graph is an extension of the cleavage graph, a data structure to reconstruct and visualize the proteolytic process. In contrast to previous approaches we extended the model to incorporate endoproteolytic processes and present a method to construct a degradation graph from mass spectrometry time series data. Based on a degradation graph and the intensities extracted from the mass spectra it is possible to estimate reaction rates of the underlying processes. We further suggest a score to rate different degradation graphs in their ability to explain the observed data. This score is used in an iterative heuristic to improve the structure of the initially constructed degradation graph.
We show that the proposed method is able to recover all degraded and generated peptides, the underlying reactions, and the reaction rates of proteolytic processes based on mass spectrometry time series data. We use simulated and real data to demonstrate that a given process can be reconstructed even in the presence of extensive noise, isobaric signals and false identifications. While the model is currently only validated on peptide data it is also applicable to proteins, as long as the necessary time series data can be produced.
We developed a decision support tool that can guide the development of heart disease prevention programs to focus on the interventions that have the most potential to benefit populations. To use it, however, users need to know the prevalence of heart disease in the population that they wish to help. We sought to determine the accuracy with which the prevalence of heart disease can be estimated from health care claims data.
We compared estimates of disease prevalence based on insurance claims to estimates derived from manual health records in a stratified random sample of 480 patients aged 30 years or older who were enrolled at any time from August 1, 2007, through July 31, 2008 (N = 474,089) in HealthPartners insurance and had a HealthPartners Medical Group electronic record. We compared randomly selected development and validation samples to a subsample that was also enrolled on August 1, 2005 (n = 272,348). We also compared the records of patients who had a gap in enrollment of more than 31 days with those who did not, and compared patients who had no visits, only 1 visit, or 2 or more visits more than 31 days apart for heart disease.
Agreement between claims data and manual review was best in both the development and the validation samples (Cohen’s κ, 0.92, 95% confidence interval [CI], 0.87–0.97; and Cohen’s κ, 0.94, 95% CI, 0.89–0.98, respectively) when patients with only 1 visit were considered to have heart disease.
In this population, prevalence of heart disease can be estimated from claims data with acceptable accuracy.
Dehydroquinate synthase (DHQS) catalyses the second step of the shikimate pathway to aromatic compounds. DHQS from the archaeal hyperthermophile Pyrococcus furiosus was insoluble when expressed in Escherichia coli but was partially solubilised when KCl was included in the cell lysis buffer. A purification procedure was developed, involving lysis by sonication at 30°C followed by a heat treatment at 70°C and anion exchange chromatography. Purified recombinant P. furiosus DHQS is a dimer with a subunit Mr of 37,397 (determined by electrospray ionisation mass spectrometry) and is active over broad pH and temperature ranges. The kinetic parameters are KM (3-deoxy-D-arabino-heptulosonate 7-phosphate) 3.7 μM and kcat 3.0 sec−1 at 60°C and pH 6.8. EDTA inactivates the enzyme, and enzyme activity is restored by several divalent metal ions including (in order of decreasing effectiveness) Cd2+, Co2+, Zn2+, and Mn2+. High activity of a DHQS in the presence of Cd2+ has not been reported for enzymes from other sources, and may be related to the bioavailability of Cd2+ for P. furiosus. This study is the first biochemical characterisation of a DHQS from a thermophilic source. Furthermore, the characterisation of this hyperthermophilic enzyme was carried out at elevated temperatures using an enzyme-coupled assay.
When waist circumference is taken into account, larger hip circumference is associated with reduced risk factors for diabetes and cardiovascular disease. The authors investigated the prospective association of hip circumference with type 2 diabetes and coronary heart disease (CHD) incidence in a biracial cohort of men and women in 4 US communities. A total of 10,767 participants from the Atherosclerosis Risk in Communities (ARIC) study were followed from 1987 to 1998. Hip and waist circumferences and body mass index (BMI) were modeled separately and mutually in association with incident diabetes and CHD by using proportional hazards regression. After adjustment for age, race, sex, and clinical center, hip circumference was positively associated with incident diabetes. However, after further controlling for waist circumference, BMI, and confounding variables, successive quintiles of hip circumference were associated with a statistically significant reduced hazard of incident diabetes (hazard ratios = 1.00, 0.79, 0.60, 0.44, 0.41). Similarly, successive quintiles of hip circumference were associated with a statistically significant reduced hazard of CHD after controlling for waist circumference, BMI, and confounding variables (hazard ratios = 1.00, 0.92, 0.75, 0.63, 0.50). Although excess adiposity is a general risk factor for diabetes and CHD, for a given BMI and waist circumference, greater hip circumference appears to lessen the risk of diabetes and CHD.
adiposity; anthropometry; coronary disease; diabetes mellitus
For at least 25 years, hypertension guidelines have suggested measuring blood pressure in both arms, but GPs' acceptance of this is low. Current and past versions of major guidelines were identified to review and assess the degree to which they provided justification, evidence, and a description of dual-arm measurement techniques. It is suggested that if guidelines better justified recommendations and cited primary literature to support claims, a greater percentage of practitioners might accept and adhere to such guidance.
blood pressure; guidelines; hypertension
As the mean age of the US population increases, the public health burden of osteoporotic fractures is expected to increase. This study prospectively examined the independent association of hip circumference with hip fracture.
The prospective association of hip circumference and hip fracture was examined in a cohort of 30,652 postmenopausal women.
Compared to the lowest quintile, successive quintiles of hip circumference were associated with a reduced hazard of hip fracture over 18 years of follow-up (HRs = 1.00, 0.78, 0.74, 0.76, 0.69, p for trend = 0.0015) after adjusting for age. Controlling for waist, this association persisted (HRs = 1.00, 0.78, 0.73, 0.72, 0.54, p for trend = 0.0006). Additionally controlling for BMI, the association of hip fracture with hip circumference was attenuated to the null while the association with successive quintiles of BMI remained significant and inverse (HRs = 1.00, 0.55, 0.45, 0.40, 0.35, p for trend <0.0001).
Although hip circumference has a strong inverse association with risk of hip fracture, this association was not independent of BMI. These results suggest that in the prediction of hip fracture risk, overall body size may be more important than body composition of the femoral-gluteal region.
anthropometry; hip fractures; cohort studies
Guidelines to prevent and treat hypertension advocate the Dietary Approaches to Stop Hypertension (DASH) diet.
We studied whether a greater concordance with the DASH diet is associated with reduced incidence of hypertension (self-reported) and mortality from cardiovascular disease in 20,993 women initially aged 55–69. We created a DASH diet concordance score using food frequency data in 1986 and followed the women for events through 2002.
No woman had perfect concordance with the DASH diet. Adjusted for age and energy intake, incidence of hypertension was inversely associated with the degree of concordance with the DASH diet, with hazard ratios across quintiles of 1.0, 0.91, 0.95, 0.99, and 0.87 (p trend = 0.02). There also were inverse, but not monotonic, associations between better DASH diet concordance and mortality from coronary heart disease, stroke, and all CVD. However, after adjustment for other risk factors, there was little evidence that any endpoint was associated with the DASH diet score.
Our results suggest that greater concordance with DASH guidelines did not have an independent long-term association with hypertension or cardiovascular mortality in this cohort. This implies that very high concordance, as achieved in the DASH trials, may be necessary to achieve the benefits of the DASH diet.
Diet; hypertension; coronary disease; cerebrovascular accident
Crystals of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from M. tuberculosis have been grown and a native data set has been collected to a maximum resolution of 2.5 Å.
The enzymes of the shikimate pathway are attractive targets for new-generation antimicrobial agents. The first step of this pathway is catalysed by 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAH7P) synthase and involves the condensation of phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) to form DAH7P. DAH7P synthases have been classified into two apparently evolutionarily unrelated types and whereas structural data have been obtained for the type I DAH7P synthases, no structural information is available for their type II counterparts. The type II DAH7P synthase from Mycobacterium tuberculosis was co-expressed as native and selenomethionine-substituted protein with the Escherichia coli chaperonins GroEL and GroES in E. coli, purified and crystallized. Native crystals of M. tuberculosis DAH7P synthase belong to space group P3121 or P3221 and diffract to 2.5 Å, with unit-cell parameters a = b = 203.61, c = 66.39 Å. There are either two or three molecules in the asymmetric unit. Multiwavelength anomalous diffraction (MAD) phasing using selenomethionine-substituted protein is currently under way.
Mycobacterium tuberculosis; shikimate pathway; chorismate biosynthesis; 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase