Spectroscopic and Computational Investigations of Ligand Binding to IspH: Discovery of Non-diphosphate Inhibitors.

Isoprenoid biosynthesis is an important area for anti-infective drug development. One isoprenoid target is (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMBPP) reductase (IspH), which forms isopentenyl diphosphate and dimethylallyl diphosphate from HMBPP in a 2H+ /2e- reduction. IspH contains a 4 Fe-4 S cluster, and in this work, we first investigated how small molecules bound to the cluster by using HYSCORE and NRVS spectroscopies. The results of these, as well as other structural and spectroscopic investigations, led to the conclusion that, in most cases, ligands bound to IspH 4 Fe-4 S clusters by η1 coordination, forming tetrahedral geometries at the unique fourth Fe, ligand side chains preventing further ligand (e.g., H2 O, O2 ) binding. Based on these ideas, we used in silico methods to find drug-like inhibitors that might occupy the HMBPP substrate binding pocket and bind to Fe, leading to the discovery of a barbituric acid analogue with a Ki value of ≈500 nm against Pseudomonas aeruginosa IspH.

3D Motions of Iron in Six-Coordinate {FeNO}(7) Hemes by Nuclear Resonance Vibration Spectroscopy.

The vibrational spectrum of a six-coordinate nitrosyl iron porphyrinate, monoclinic [Fe(TpFPP)(1-MeIm)(NO)] (TpFPP=tetra-para-fluorophenylporphyrin; 1-MeIm=1-methylimidazole), has been studied by oriented single-crystal nuclear resonance vibrational spectroscopy (NRVS). The crystal was oriented to give spectra perpendicular to the porphyrin plane and two in-plane spectra perpendicular or parallel to the projection of the FeNO plane. These enable assignment of the FeNO bending and stretching modes. The measurements reveal that the two in-plane spectra have substantial differences that result from the strongly bonded axial NO ligand. The direction of the in-plane iron motion is found to be largely parallel and perpendicular to the projection of the bent FeNO on the porphyrin plane. The out-of-plane Fe-N-O stretching and bending modes are strongly mixed with each other, as well as with porphyrin ligand modes. The stretch is mixed with v50 as was also observed for dioxygen complexes. The frequency of the assigned stretching mode of eight Fe-X-O (X=N, C, and O) complexes is correlated with the Fe-XO bond lengths. The nature of highest frequency band at ≈560 cm(-1) has also been examined in two additional new derivatives. Previously assigned as the Fe-NO stretch (by resonance Raman), it is better described as the bend, as the motion of the central nitrogen atom of the FeNO group is very large. There is significant mixing of this mode. The results emphasize the importance of mode mixing; the extent of mixing must be related to the peripheral phenyl substituents.

A strenuous experimental journey searching for spectroscopic evidence of a bridging nickel-iron-hydride in [NiFe] hydrogenase.

Direct spectroscopic evidence for a hydride bridge in the Ni-R form of [NiFe] hydrogenase has been obtained using iron-specific nuclear resonance vibrational spectroscopy (NRVS). The Ni-H-Fe wag mode at 675 cm(-1) is the first spectroscopic evidence for a bridging hydride in Ni-R as well as the first iron-hydride-related NRVS feature observed for a biological system. Although density function theory (DFT) calculation assisted the determination of the Ni-R structure, it did not predict the Ni-H-Fe wag mode at ∼ 675 cm(-1) before NRVS. Instead, the observed Ni-H-Fe mode provided a critical reference for the DFT calculations. While the overall science about Ni-R is presented and discussed elsewhere, this article focuses on the long and strenuous experimental journey to search for and experimentally identify the Ni-H-Fe wag mode in a Ni-R sample. As a methodology, the results presented here will go beyond Ni-R and hydrogenase research and will also be of interest to other scientists who use synchrotron radiation for measuring dilute samples or weak spectroscopic features.

Assessing the Nutrient Status of Low Carbohydrate, High-Fat (LCHF) Meal Plans in Children: A Hypothetical Case Study Design.

There is well-established evidence for low-carbohydrate, high-fat (LCHF) diets in the management of chronic health conditions in adults. The natural next step is to understand the potential risks and benefits of LCHF diets for children, where they may have useful applications for general health and a variety of chronic health conditions. It is vital that any diet delivers sufficient micronutrients and energy to ensure health, wellbeing, and growth. This descriptive study assesses the nutrient and energy status of LCHF sample meal plans for children. We designed four meal plans for hypothetical weight-stable male and female children (11 years) and adolescents (16 years). Carbohydrates were limited to ≤80 g, protein was set at 15-25% of the total energy, and fat supplied the remaining calories. Using FoodWorks dietary analysis software, data were assessed against the national Australian/New Zealand nutrient reference value (NRV) thresholds for children and adolescents. All meal plans exceeded the minimum NRV thresholds for all micronutrients; protein slightly exceeded the AMDR recommendations by up to three percentage points. This study demonstrates that LCHF meal plans can be energy-, protein-, and micronutrient-replete for children and adolescents. As with any dietary approach, well-formulated meals and careful planning are key to achieving the optimal nutrient status.

Reconsidering the Tolerable Upper Levels of Zinc Intake among Infants and Young Children: A Systematic Review of the Available Evidence.

Safe upper levels (UL) of zinc intake for children were established based on either (1) limited data from just one study among children or (2) extrapolations from studies in adults. Resulting ULs are less than amounts of zinc consumed by children in many studies that reported benefits of zinc interventions, and usual dietary zinc intakes often exceed the UL, with no apparent adverse effects. Therefore, existing ULs may be too low. We conducted a systematic bibliographic review of studies among preadolescent children, in which (1) additional zinc was provided vs. no additional zinc provided, and (2) the effect of zinc on serum or plasma copper, ceruloplasmin, ferritin, transferrin receptor, lipids, or hemoglobin or erythrocyte super-oxide dismutase were assessed. We extracted data from 44 relevant studies with 141 comparisons. Meta-analyses found no significant overall effect of providing additional zinc, except for a significant negative effect on ferritin (p = 0.001), albeit not consistent in relation to the zinc dose. Interpretation is complicated by the significant heterogeneity of results and uncertainties regarding the physiological and clinical significance of outcomes. Current zinc ULs should be reassessed and potentially revised using data now available for preadolescent children and considering challenges regarding interpretation of results.

Nuclear Resonance Vibrational Spectroscopy: A Modern Tool to Pinpoint Site-Specific Cooperative Processes.

Nuclear resonant vibrational spectroscopy (NRVS) is a synchrotron radiation (SR)-based nuclear inelastic scattering spectroscopy that measures the phonons (i.e., vibrational modes) associated with the nuclear transition. It has distinct advantages over traditional vibration spectroscopy and has wide applications in physics, chemistry, bioinorganic chemistry, materials sciences, and geology, as well as many other research areas. In this article, we present a scientific and figurative description of this yet modern tool for the potential users in various research fields in the future. In addition to short discussions on its development history, principles, and other theoretical issues, the focus of this article is on the experimental aspects, such as the instruments, the practical measurement issues, the data process, and a few examples of its applications. The article concludes with introduction to non-57Fe NRVS and an outlook on the impact from the future upgrade of SR rings.

Overweight & obese Australian adults and micronutrient deficiency.

BACKGROUND: Micronutrients have been implicated as an important factor in regulating various metabolic processes and thus playing a role in the aetiology of obesity. Many studies have been conducted worldwide that clearly show a direct link between obesity and micronutrient deficiencies. The aim of this study was to assess the nutritional status of overweight and obese Australian adults to see if there were any associations between BMI and serum micronutrient levels. METHODS: Baseline serum micronutrient data of overweight and obese individuals with a body mass index (BMI) between 25 and 40 kg/m2 and aged between 18 and 65 years was compared to the clinical micronutrient reference ranges for associations between BMI and micronutrient status. RESULTS: There were significant negative associations between BMI and serum vitamin D (p = 0.044), folate (p = 0.025), magnesium (p = 0.010) and potassium (p = 0.023). CONCLUSIONS: Overweight and obesity appears to impact on the bioavailability and utilisation of micronutrients with absorption, excretion, storage/distribution (fat sequestering, tissue dispersion), metabolism (catabolic losses, possibly oxidative), increased physiologic requirements, and lower absolute total dietary intake being the current theory for observed differences. While vitamins D, folate, magnesium and potassium showed a negative relationship to BMI, other micronutrients did not. This may be explained by the fortification of certain processed foods, or the possibility of overweight and obese people eating more to satisfy their nutritional requirements.

Assessing the nutrient intake of a low-carbohydrate, high-fat (LCHF) diet: a hypothetical case study design.

OBJECTIVE: The low-carbohydrate, high-fat (LCHF) diet is becoming increasingly employed in clinical dietetic practice as a means to manage many health-related conditions. Yet, it continues to remain contentious in nutrition circles due to a belief that the diet is devoid of nutrients and concern around its saturated fat content. This work aimed to assess the micronutrient intake of the LCHF diet under two conditions of saturated fat thresholds. DESIGN: In this descriptive study, two LCHF meal plans were designed for two hypothetical cases representing the average Australian male and female weight-stable adult. National documented heights, a body mass index of 22.5 to establish weight and a 1.6 activity factor were used to estimate total energy intake using the Schofield equation. Carbohydrate was limited to <130 g, protein was set at 15%-25% of total energy and fat supplied the remaining calories. One version of the diet aligned with the national saturated fat guideline threshold of <10% of total energy and the other included saturated fat ad libitum. PRIMARY OUTCOMES: The primary outcomes included all micronutrients, which were assessed using FoodWorks dietary analysis software against national Australian/New Zealand nutrient reference value (NRV) thresholds. RESULTS: All of the meal plans exceeded the minimum NRV thresholds, apart from iron in the female meal plans, which achieved 86%-98% of the threshold. Saturated fat intake was logistically unable to be reduced below the 10% threshold for the male plan but exceeded the threshold by 2 g (0.6%). CONCLUSION: Despite macronutrient proportions not aligning with current national dietary guidelines, a well-planned LCHF meal plan can be considered micronutrient replete. This is an important finding for health professionals, consumers and critics of LCHF nutrition, as it dispels the myth that these diets are suboptimal in their micronutrient supply. As with any diet, for optimal nutrient achievement, meals need to be well formulated.

NRVS for Fe in Biology: Experiment and Basic Interpretation.

For over 20 years, nuclear resonance vibrational spectroscopy (NRVS) has been used to study vibrational dynamics of iron-containing materials. With the only selection rule being iron motion, 57Fe NRVS has become an excellent tool to study iron-containing enzymes. Over the past decade, considerable progress has been made in the study of complex metalloenzymes using NRVS. Iron cofactors in heme-containing globins; [2Fe2S], [3Fe4S], [4Fe4S] proteins; the [NiFe] and [FeFe] hydrogenases; and nitrogenases have been explored in a fashion not possible through traditional vibrational spectroscopy. In this chapter, we discuss the basics of NRVS, a strategy to perform NRVS, and a discussion of the application of NRVS on rubredoxin and [FeFe] hydrogenase.

Proton Transfer in the Catalytic Cycle of [NiFe] Hydrogenases: Insight from Vibrational Spectroscopy.

Catalysis of H2 production and oxidation reactions is critical in renewable energy systems based around H2 as a clean fuel, but the present reliance on platinum-based catalysts is not sustainable. In nature, H2 is oxidized at minimal overpotential and high turnover frequencies at [NiFe] catalytic sites in hydrogenase enzymes. Although an outline mechanism has been established for the [NiFe] hydrogenases involving heterolytic cleavage of H2 followed by a first and then second transfer of a proton and electron away from the active site, details remain vague concerning how the proton transfers are facilitated by the protein environment close to the active site. Furthermore, although [NiFe] hydrogenases from different organisms or cellular environments share a common active site, they exhibit a broad range of catalytic characteristics indicating the importance of subtle changes in the surrounding protein in controlling their behavior. Here we review recent time-resolved infrared (IR) spectroscopic studies and IR spectroelectrochemical studies carried out in situ during electrocatalytic turnover. Additionally, we re-evaluate the significant body of IR spectroscopic data on hydrogenase active site states determined through more conventional solution studies, in order to highlight mechanistic steps that seem to apply generally across the [NiFe] hydrogenases, as well as steps which so far seem limited to specific groups of these enzymes. This analysis is intended to help focus attention on the key open questions where further work is needed to assess important aspects of proton and electron transfer in the mechanism of [NiFe] hydrogenases.

Low frequency dynamics of the nitrogenase MoFe protein via femtosecond pump probe spectroscopy - Observation of a candidate promoting vibration.

We have used femtosecond pump-probe spectroscopy (FPPS) to study the FeMo-cofactor within the nitrogenase (N2ase) MoFe protein from Azotobacter vinelandii. A sub-20-fs visible laser pulse was used to pump the sample to an excited electronic state, and a second sub-10-fs pulse was used to probe changes in transmission as a function of probe wavelength and delay time. The excited protein relaxes to the ground state with a ~1.2ps time constant. With the short laser pulse we coherently excited the vibrational modes associated with the FeMo-cofactor active site, which are then observed in the time domain. Superimposed on the relaxation dynamics, we distinguished a variety of oscillation frequencies with the strongest band peaks at ~84, 116, 189, and 226cm(-1). Comparison with data from nuclear resonance vibrational spectroscopy (NRVS) shows that the latter pair of signals comes predominantly from the FeMo-cofactor. The frequencies obtained from the FPPS experiment were interpreted with normal mode calculations using both an empirical force field (EFF) and density functional theory (DFT). The FPPS data were also compared with the first reported resonance Raman (RR) spectrum of the N2ase MoFe protein. This approach allows us to outline and assign vibrational modes having relevance to the catalytic activity of N2ase. In particular, the 226cm(-1) band is assigned as a potential 'promoting vibration' in the H-atom transfer (or proton-coupled electron transfer) processes that are an essential feature of N2ase catalysis. The results demonstrate that high-quality room-temperature solution data can be obtained on the MoFe protein by the FPPS technique and that these data provide added insight to the motions and possible operation of this protein and its catalytic prosthetic group.

Nuclear resonance vibrational spectroscopy (NRVS) of rubredoxin and MoFe protein crystals.

We have applied 57Fe nuclear resonance vibrational spectroscopy (NRVS) for the first time to study the dynamics of Fe centers in Fe-S protein crystals, including oxidized wild type rubredoxin crystals from Pyrococcus furiosus, and the MoFe protein of nitrogenase from Azotobacter vinelandii. Thanks to the NRVS selection rule, selectively probed vibrational modes have been observed in both oriented rubredoxin and MoFe protein crystals. The NRVS work was complemented by extended X-ray absorption fine structure spectroscopy (EXAFS) measurements on oxidized wild type rubredoxin crystals from Pyrococcus furiosus. The EXAFS spectra revealed the Fe-S bond length difference in oxidized Pf Rd protein, which is qualitatively consistent with the X-ray crystal structure.

Application of Dietary Reference Intakes for Codex Nutrient Reference Values

Codex Nutrient Reference Values (NRVs) were based on the 1988 Helsinki report and a single set of NRVs was established for the general population over 3 years for the purpose of food labeling in 1993. Dietary Reference Intakes (DRIs) with new concepts were developed in many countries recently. Hence there has been broad discussion regarding establishment of new NRVs using DRIs, which include estimated average requirement (EAR), recommended intake (RI or RDA), adequate intake (AI), and tolerable upper intake level (UL) in the world. This study was carried out to review various possible values for Codex NRVs such as population-weighted EAR, population-weighted RDA, and population-based RDA. The values were simulated using DRIs and population distribution of Korea, USA, Japan, Philippines, Germany and England, and compared to the current Codex values and the highest RDA and the lowest UL among populations with different life-stage. Since population weighted/based values are necessary to be updated according to the population changes and are different across countries, inconsistency can be a serious barrier in international transactions. For some of nutrients such as vitamin A and zinc, values based on population-based RDA or the highest RDA were higher than the lowest UL. Therefore, careful considerations should be given before establishing Codex NRVs.

Scientific Principles on the Revision and Addition of the Codex Nutrient Reference Values for Food Labelling Purposes

There has been a need to update the current Codex nutrient reference values (NRVs) which had been established following the Helsinki Consultation in 1988. The main purposes of NRVs are to help consumers compare the nutrient content of different food products and estimate the usefulness of a food product in terms of its percentage contribution to the overall healthful diets. The establishment of Codex NRVs for labelling purposes is intended to facilitate the goals of protecting consumers' health and ensuring fair practices in food trade. When revising the Codex NRVs several issues are needed to discussed, which are: 1) Consideration of population groups whether to have one set of NRVs for general population or to have several sets of NRVs for each population groups, 2) Determination of scope of nutrients such as what nutrients to include and what reference values to choose and 3) Development of principles for establishing NRVs such as the selection of suitable reference values among many including recommended intake/recommended dietary allowances, estimated average requirement and upper levels to extract NRVs. This paper will discuss the above issues and the inputs and efforts that the Korean Nutrition Society had put into to carry out the project, "International Collaboration on the Revision and Addition of the Codex Nutrient Reference Values for Food Labelling Purposes," and to ascertain relevant scientific principles and evidences that are utilized for establishment and revision of the Codex NRVs.

The Scope of Population Groups and Nutrients for Codex Nutrient Reference Values

The purposes of this study were to establish the scope of population groups to be set and nutrients to be included for Codex Nutrient Reference Values (NRVs). Examination of a variety of documents and reports on the principles of scientific evidence was gone through to choose representative groups from a variety of population groups and kinds of nutrients for setting Codex NRVs. Also, hearings from the groups of NRVs expertise has been formed and was gone through by e-working process. The differences of food and foods being consumed in general and by specific population groups and limited actual space on packages for food labeling were considered. This paper suggest the options on the scope of population groups to be set and nutrients to be included for Codex NRVs on the basis of the above procedures. Also, the advantages and disadvantages of these options are commented when the options are applied for establishment and revision of the Codex NRVs.