Phosphate forms an unusual tripodal complex with the Fe-Mn center of sweet potato purple acid phosphatase

Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)-Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a mu-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (k(cat)/K-m) at pH 4.5, whereas its catalytic rate constant (k(cat)) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pK(a) of the leaving group. The crystal structure of the phosphate-bound Fe(III)-Mn(II) PAP has been determined to 2.5-Angstrom resolution (final R-free value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to A protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis.

The crystal structure of a bacterial Class II ketol-acid reductolsomerase: Domain conservation and evolution

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes two steps in the biosynthesis of branched-chain amino acids. Amino acid sequence comparisons across species reveal that there are two types of this enzyme: a short form (Class 1) found in fungi and most bacteria, and a long form (Class 11) typical of plants. Crystal structures of each have been reported previously. However, some bacteria such as Escherichia coli possess a long form, where the amino acid sequence differs appreciably from that found in plants. Here, we report the crystal structure of the E. coli enzyme at 2.6 A resolution, the first three-dimensional structure of any bacterial Class 11 KARI. The enzyme consists of two domains, one with mixed alpha/beta structure, which is similar to that found in other pyridine nucleotide-dependent dehydrogenases. The second domain is mainly alpha-helical and shows strong evidence of internal duplication. Comparison of the active sites between KARI of E. coli, Pseudomonas aeruginosa, and spinach shows that most residues occupy conserved positions in the active site. E. coli KARI was crystallized as a tetramer, the likely biologically active unit. This contrasts with P. aeruginosa KARI, which forms a dodecamer, and spinach KARI, a dimer. In the E. coli KARI tetramer, a novel subunit-to-subunit interacting surface is formed by a symmetrical pair of bulbous protrusions.

Apparent ileal digestibility of amino acids in dietary ingredients for broiler chickens

The apparent ileal digestibility coefficients of amino acids in 107 samples representing 22 food ingredients were determined using 6-week-old broiler chickens. The ingredients assayed included five cereals ( barley, maize, sorghum, triticale and wheat), two cereal by-products ( rice polishings and wheat middlings), four oilseed meals ( canola, cottonseed, soyabean and sunflower meals), full-fat canola, maize gluten meal, four grain legumes ( chickpeas, faba beans,field peas and lupins) and five animal protein sources ( blood, feather,fish, meat and meat and bone meals). The mean ileal digestibility coefficients of amino acids in wheat and maize were higher than those in sorghum, triticale and barley. However, variations observed in individual amino acid digestibilities among samples within cereal type were greater than those determined between cereals. Threonine and lysine were the least digestible indispensable amino acids in the five cereals evaluated. The most digestible indispensable amino acid was phenylalanine in wheat and, leucine in maize and sorghum. In the case of the wheat middlings and rice polishings, threonine was the least digestible indispensable amino acid and arginine was the best digested. In the oilseed meals assayed, amino acid digestibility was highest for soya-bean and sunflower meals, intermediate for canola meal and lowest for cottonseed meal. Ileal digestibility coefficients of amino acids in lupins were found to be slightly lower than those in soya-bean meal. The amino acid digestibilities of field peas, faba beans and chickpeas were considerably lower than those of lupins. Digestibility of arginine was the highest and that of threonine was the lowest of the indispensable amino acids in oilseed meals and grain legumes, except in cottonseed meal. Lysine was the least digestible amino acid in cottonseed meal. In the animal protein sources assayed, digestibility coefficients of amino acids in blood meal were high, intermediate in fish meal, and low in meat meal, meat and bone meal and feather meal. Variation in amino acid digestibility coefficients determined for blood meal samples was small. However, wide variations in amino acid digestibilities were observed for other animal protein sources, highlighting significant batch-to-batch differences. In particular, marked variations were determined for meat meal and meat and bone meal samples. Cystine was the least digested amino acid in animal protein meals, with the exception of blood meal in which isoleucine had the lowest digestibility. The limitations of using apparent digestibility values in diet formulations and the concept of the standardized digestibility system to overcome these limitations are discussed.

Formulation and characterisation of lyophilised rapid disintegrating tablets using amino acids as matrix forming agents

Despite recent advances in the formulation of lyophilised rapid disintegrating tablets (RDTs), the inclusion of matrix supporting/disintegration enhancing agents has been limited to the use of saccharides and polyols. In this study, the feasibility of using amino acids as matrix forming agents in lyophilised RDTs was investigated. Twelve amino acids were chosen (alanine, arginine, threonine, glycine, cysteine, serine, histidine, lysine, valine, asparagine, glutamine and proline), and the suitability for freeze drying, mechanical properties and disintegration time after inclusion of the amino acids at varied concentration were studied. In addition, the porosity of the RDTs and wettability profile of the amino acids were investigated to understand the mechanisms of disintegration. The results suggest the suitability of these amino acids for the lyophilisation regime, as they displayed satisfactory safety margin between the glass transition and shelf temperature (-40 degrees C), except proline-based formulations. Moreover, the crystallisation behavior of alanine, glycine, cysteine and serine at high concentration increased the stability of the formulation. The characterisation of the RDTs suggests that high concentration of the amino acids is required to enhance the mechanical properties, whereas only optimum concentrations promote the disintegration. Moreover, wetting time of the amino acid and porosity of the tablet are the two factors that control the disintegration of RDTs.

The development of a model system and high throughput assay for the study of interactions between zinc finger proteins and DNA

It has been recognised for some time that a full code of amino acid-based recognition of DNA sequences would be useful. Several approaches, which utilise small DNA binding motifs called zinc fingers, are presently employed. None of the current approaches successfully combine a combinatorial approach to the elucidation of a code with a single stage high throughput screening assay. The work outlined here describes the development of a model system for the study of DNA protein interactions and the development of a high throughput assay for detection of such interactions. A zinc finger protein was designed which will bind with high affinity and specificity to a known DNA sequence. For future work it is possible to mutate the region of the zinc finger responsible for the specificity of binding, in order to observe the effect on the DNA / protein interactions. The zinc finger protein was initially synthesised as a His tagged product. It was not possible however to develop a high throughput assay using the His tagged zinc finger protein. The gene encoding the zinc finger protein was altered and the protein synthesised as a Glutathione S-Transferase (GST) fusion product. A successful assay was developed using the GST protein and Scintillation Proximity Assay technology (Amersham Pharmacia Biotech). The scintillation proximity assay is a dynamic assay that allows the DNA protein interactions to be studied in "real time". This assay not only provides a high throughput method of screening zinc finger proteins for potential ligands but also allows the effect of addition of reagents or competitor ligands to be monitored.

Multiple roles of charged amino acids in cytoplasmic loop 7 for expression and function of the multidrug and organic anion transporter MRP1 (ABCC1)

Multidrug resistance protein MRP1 mediates the ATP-dependent efflux of many chemotherapeutic agents and organic anions. MRP1 has two nucleotide binding sites (NBSs) and three membrane spanning domains (MSDs) containing 17 transmembrane helices linked by extracellular and cytoplasmic loops (CL). Homology models suggest that CL7 (amino acids 1141-1195) is in a position where it could participate in signaling between the MSDs and NBSs during the transport process. We have individually replaced eight charged residues in CL7 with Ala, and in some cases, an amino acid with the same charge, and then investigated the effects on MRP1 expression, transport activity, and nucleotide and substrate interactions. A triple mutant in which Glu(1169), Glu(1170), and Glu(1172) were all replaced with Ala was also examined. The properties of R1173A and E1184A were comparable with those of wild-type MRP1, whereas the remaining mutants were either poorly expressed (R1166A, D1183A) or exhibited reduced transport of one or more organic anions (E1144A, D1179A, K1181A, (1169)AAQA). Same charge mutant D1183E was also not expressed, whereas expression and activity of R1166K were similar to wild-type MRP1. The moderate substrate-selective changes in transport activity displayed by mutants E1144A, D1179A, K1181A, and (1169)AAQA were accompanied by changes in orthovanadate-induced trapping of [alpha-(32)P]azidoADP by NBS2 indicating changes in ATP hydrolysis or release of ADP. In the case of E1144A, estradiol glucuronide no longer inhibited trapping of azidoADP. Together, our results demonstrate the extreme sensitivity of CL7 to mutation, consistent with its critical and complex dual role in both the proper folding and transport activity of MRP1.

ProxiMAX Randomisation:a new technology for non-degenerate saturation mutagenesis of contiguous codons

ProxiMAX randomisation achieves saturation mutagenesis of contiguous codons without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, it uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents and as such, requires no specialised chemistry, reagents nor equipment. When particular residues are known to affect protein activity/specificity, their combinatorial replacement with all 20 amino acids, or a subset thereof, can provide a rapid route to generating proteins with desirable characteristics. Conventionally, saturation mutagenesis replaced key codons with degenerate ones. Although simple to perform, that procedure resulted in unnecessarily large libraries, termination codons and inherent uneven amino acid representation. ProxiMAX randomisation is an enzyme-based technique that can encode unbiased representation of all or selected amino acids or else can provide required codons in pre-defined ratios. Each saturated position can be defined independently of the others. ProxiMAX randomisation is achieved via saturation cycling: an iterative process comprising blunt end ligation, amplification and digestion with a Type IIS restriction enzyme. We demonstrate both unbiased saturation of a short 6-mer peptide and saturation of a hypervariable region of a scfv antibody fragment, where 11 contiguous codons are saturated with selected codons, in pre-defined ratios. As such, ProxiMAX randomisation is particularly relevant to antibody engineering. The development of ProxiMAX randomisation from concept to reality is described.

Covalent protein adduction of nitrogen mustards and related compounds

Chemical warfare agents continue to pose a global threat despite the efforts of the international community to prohibit their use in warfare. For this reason, improvement in the detection of these compounds remains of forensic interest. Protein adducts formed by the covalent modification of an electrophilic xenobiotic and a nucleophilic amino acid may provide a biomarker of exposure that is stable and specific to compounds of interest (such as chemical warfare agents), and have the capability to extend the window of detection further than the parent compound or circulating metabolites. This research investigated the formation of protein adducts of the nitrogen mustard chemical warfare agents mechlorethamine (HN-2) and tris(2-chloroethyl)amine (HN-3) to lysine and histidine residues found on the blood proteins hemoglobin and human serum albumin. Identified adducts were assessed for reproducibility and stability both in model peptide and whole protein assays. Specificity of these identified adducts was assessed using in vitro assays to metabolize common therapeutic drugs containing nitrogen mustard moieties. Results of the model peptide assays demonstrated that HN-2 and HN-3 were able to form stable adducts with lysine and histidine residues under physiological conditions. Results for whole protein assays identified three histidine adducts on hemoglobin, and three adducts (two lysine residues and one histidine residue) on human serum albumin that were previously unknown. These protein adducts were determined to be reproducible and stable at physiological conditions over a three-week analysis period. Results from the in vitro metabolic assays revealed that adducts formed by HN-2 and HN-3 are specific to these agents, as metabolized therapeutic drugs (chlorambucil, cyclophosphamide, and melphalan) did not form the same adducts on lysine or histidine residues as the previously identified adducts formed by HN-2 and HN-3. Results obtained from the model peptide and full protein work were enhanced by comparing experimental data to theoretical calculations for adduct formation, providing further confirmatory data. This project was successful in identifying and characterizing biomarkers of exposure to HN-2 and HN-3 that are specific and stable and which have the potential to be used for the forensic determination of exposure to these dangerous agents.

Experimental and Theoretical Models to Probe Mechanisms of Biological Charge Flow

Nature is challenged to move charge efficiently over many length scales. From sub-nm to μm distances, electron-transfer proteins orchestrate energy conversion, storage, and release both inside and outside the cell. Uncovering the detailed mechanisms of biological electron-transfer reactions, which are often coupled to bond-breaking and bond-making events, is essential to designing durable, artificial energy conversion systems that mimic the specificity and efficiency of their natural counterparts. Here, we use theoretical modeling of long-distance charge hopping (Chapter 3), synthetic donor-bridge-acceptor molecules (Chapters 4, 5, and 6), and de novo protein design (Chapters 5 and 6) to investigate general principles that govern light-driven and electrochemically driven electron-transfer reactions in biology. We show that fast, μm-distance charge hopping along bacterial nanowires requires closely packed charge carriers with low reorganization energies (Chapter 3); singlet excited-state electronic polarization of supermolecular electron donors can attenuate intersystem crossing yields to lower-energy, oppositely polarized, donor triplet states (Chapter 4); the effective static dielectric constant of a small (~100 residue) de novo designed 4-helical protein bundle can change upon phototriggering an electron transfer event in the protein interior, providing a means to slow the charge-recombination reaction (Chapter 5); and a tightly-packed de novo designed 4-helix protein bundle can drastically alter charge-transfer driving forces of photo-induced amino acid radical formation in the bundle interior, effectively turning off a light-driven oxidation reaction that occurs in organic solvent (Chapter 6). This work leverages unique insights gleaned from proteins designed from scratch that bind synthetic donor-bridge-acceptor molecules that can also be studied in organic solvents, opening new avenues of exploration into the factors critical for protein control of charge flow in biology.

(Table 2) Concentrations and D/L values for three amino acids in bulk foraminifera from ODP Sites 182-1126 and 182-1127

A number of studies have shown that methanogens are active in the presence of sulfate under some conditions. This phenomenon is especially exemplified in carbonate sediments of the southern Australian continental margin. Three sites cored during Ocean Drilling Program (ODP) Leg 182 in the Great Australian Bight have high concentrations of microbially-generated methane and hydrogen sulfide throughout almost 500 m of sediments. In these cores, the sulfate-reducing and methanogenic zones overlap completely; that is, the usual sulfate-methane transition zone is absent. Amino acid racemization data show that the gassy sediments consist of younger carbonates than the low-gas sites. High concentrations of the reduced gases also occur in two ODP sites on the margin of the Bahamas platform, both of which have similar sedimentary conditions to those of the high-gas sites of Leg 182. Co-generation of these reduced gases results from an unusual combination of conditions, including: (1) a thick Quaternary sequence of iron-poor carbonate sediments, (2) a sub-seafloor brine, and (3) moderate amounts of organic carbon. The probable explanation for the co-generation of hydrogen sulfide and methane in all these sites, as well as in other reported environments, is that methanogens are utilizing non-competitive substrates to produce methane within the sulfate-reducing zone. Taken together, these results form the basis of a new model for sulfate reduction and methanogenesis in marine sediments. The biogeochemical end-members of the model are: (1) minimal sulfate reduction, (2) complete sulfate reduction followed by methanogenesis, and (3) overlapping sulfate reduction and methanogenesis with no transition zone.

Granulometry, geochemistry, amino acids, radiocarbon ages and paleomagnetics of samples from Heidemann Valley

A Pliocene (2.6-3.5 Ma) age is determined from glacial sediments studied in a 20m long, 4 m deep trench excavated in Heidemann Valley, Vestfold Hills, East Antarctica. The age determination is based on a combined study of amino acid racemization, diatoms, foraminifera, and magnetic polarity, and supports earlier estimates of the age of the sedimentary section; all are beyond 14C range. Four till units are recognized and documented, and 16 subunits are identified. All are ascribed to deposition during a Late Pliocene glaciation that was probably the last time the entire Vestfold Hills was covered by an enlarged East Antarctic Ice Sheet (EAIS). Evidence for other more recent glacial events of the 'Vestfold Glaciation' may have been due to lateral expansion of the Sorsdal Glacier and limited expansion of the icesheet margin during the Last Glacial Maximum rather than a major expansion of the EAIS. The deposit appears to correlate with a marine deposition event recorded in Ocean Drilling Program Site 1166 in Prydz Bay, possibly with the Bardin Bluffs Formation of the Prince Charles Mountains and with part of the time represented in the ANDRILL AND-1B core in the Ross Sea.

(Table 1) Concentrations of amino acids in sediments from DSDP Site 68-502

In this chapter, we will report on the amino acids in the total acid hydrolysate of eight sediment samples from Leg 68 Site 502. This site was located on a topographic high at a depth of 3051 meters in the Colombian Basin of the western Caribbean Sea. Four holes were cored at the site by means of the hydraulic piston corer to a maximum sediment depth of 218 meters. The composite section is a virtually continuous, undisturbed sediment record covering almost 8 million years from the Holocene to late Miocene. Age estimates for the section are based on excellent magnetostratigraphic and biostratigraphic records. Four lithostratigraphic units (A, B, C, and D) were recognized, based on differences in color and content of clay, ash, foraminifers, and siliceous microfossils (Prell, Gardner, et al., 1980): A, yellowish brown to light brownish gray foraminifer-bearing (> 10%) nannofossil marl; B, gray to olive gray foraminifer-bearing nannofossil marl with occasional ash beds; C, light gray to dark greenish gray calcareous clay and foraminifer-bearing (< 10%) nannofossil marl; D, pale green to grayish green calcareous, ash-bearing clay with siliceous microfossils. The calcium carbonate content of these sediments increases from about 27 to about 49% from late Miocene to middle Pliocene (about 3.6 Ma) and remains uniform at about 48 to 50% from that time throughout the Quaternary. The eight sediment samples for amino acid analyses came from the third (502B) and fourth (502C) holes at Site 502. Samples ranged in sub-bottom depth from 4.3 to 225 meters spanning time from 0.3 to 7.7 Ma.

Functions of the SLC36 transporter Pathetic in growth control of Drosophila sensory neurons

Thesis (Ph.D.)--University of Washington, 2016-08