Using D- and L-Amino Acid Oxidases to Generate the Imino Acid Substrate to Measure the Activity of the Novel Rid (Enamine/Imine Deaminase) Class of Enzymes.

This chapter describes a method to assay the activity of reactive intermediate deaminases (Rid), a large family of conserved soluble enzymes, which have been proposed to prevent damages from metabolic intermediates such as the highly reactive and unstable compounds enamines/imines. In this method, the flavin adenine dinucleotide-dependent L- or D-amino acid oxidases generate an imino acid starting from a L- or D- amino acid, respectively. This reaction is coupled to the hydrolysis of the imino acid to the corresponding α-keto acid and ammonium ion catalyzed by a Rid enzyme. The spectrophotometric assay consists of measuring the decrease of the initial rate of formation of the semicarbazone, derived from the spontaneous reaction of the imino acid and semicarbazide, caused by the presence of the Rid enzyme. The set-up and testing of this method imply a preliminary characterization of the ability of the amino acid oxidase to release the imino acid required for the subsequent reactions. To this purpose, the activity of the L- or D-amino acid oxidases with different amino acids can be measured as production of hydrogen peroxide or formation of semicarbazone in parallel assays. The advantages and limitations of this assay of Rid activity are discussed.

An Asymmetric Reductase That Intercepts Acyclic Imino Acids Produced in Situ by a Partner Oxidase.

Acyclic imines are unstable in aqueous conditions. For this reason, known imine reductases, which enable the synthesis of chiral amines, mainly intercept stable cyclic imines. Here we report the detailed biochemical and structural characterization of Bsp5, an imino acid reductase from the d-2-hydroxyacid dehydrogenase family that reduces acyclic imino acids produced in situ by a partner oxidase. We determine a 1.6 Å resolution structure of Bsp5 in complex with d-arginine and coenzyme NADPH. Combined with mutagenesis work, our study reveals the minimal structural constraints for its biosynthetic activity. Furthermore, we demonstrate that Bsp5 can intercept more complex products from an alternate oxidase partner, suggesting that this oxidase-imino acid reductase pair could be evolved for biocatalytic conversion of l-amino acids to d-amino acids.

Synthesis and bioevaluation of technetium-99 m / rhenium labeled phenylquinoxaline derivatives as Tau imaging probes.

Based on our previous research on the fluorinated phenylquinoxaline scaffold, in this study, different positions of N,N-dimethyl amino group, and alkyl linkers with various lengths were introduced into this scaffold to regulate their lipophilicity and binding affinity to Tau. Four novel 99mTc/Re complexes with diethyl iminodiacetate chelator were synthesized and evaluated as Tau imaging tracers in the brain of Alzheimer's disease. Their specific binding to neurofibrillary tangles was verified by in vitro fluorescence staining and further confirmed by the results of immunofluorescence staining on the same brain sections from AD patient and Tg-tau mice. From in vitro binding assay using recombinant Tau aggregates, complex 4.2 with 6-N(CH3)2 and longer carbon chain (n = 4) displayed the highest affinity (Kd = 59.95 nM). [99mTc]4.2 was achieved by the ligand exchange reaction between dicarboxylic precursor and [99mTc(CO)3(H2O)3]+ intermediate with radiochemical yield over 45%. Ex vivo biodistribution studies on normal ICR mice revealed that [99mTc]4.2 exhibited moderate initial brain uptake (0.61% ID/g) and more structure optimizations are still required to improve the blood-brain barrier permeability.

LC-MS/MS reveals the formation of reactive ortho-quinone and iminium intermediates in saracatinib metabolism: Phase I metabolic profiling.

Saracatinib (AZD-0530) is a drug under clinical trials that developed by AstraZeneca. It is considered a dual kinase inhibitor, with selective actions as a Src inhibitor and a Bcr-Abl tyrosine-kinase inhibitor. Saracatinib chemical structure contains N-methyl piperazine group and 1,3 benzodioxole group. N-methyl piperazine group that can be bioactivated to form iminium intermediates which can be captured by KCN. 1,3-Benzodioxole group can be bioactivated to form ortho-quinone intermediate that can be conjugated with GSH. The formed conjugates are stable and can be identified using LC-MS/MS. In our current work, we are trying to give insight into the reasons that may be responsible for saracatinib side effects. Using LC-MS/MS, in vitro metabolic pathways were investigated for saracatinib in rat liver microsomes. Ten saracatinib phase I metabolites were characterized and the metabolic pathways were found to be hydroxylation, oxidation, reduction, dealkylation, N-oxidation and ether cleavage. Also, four potential reactive intermediates (three cyanide adducts and one GSH conjugate) were identified and the bioactivation mechanisms were explained. The existence of these four reactive metabolites may be the main reason for observed saracatinib side effects in clinical trials. Literature review showed no previous articles have been proposed the detailed structural identification of the formed reactive metabolites.

Crystal structure of the 2-iminoglutarate-bound complex of glutamate dehydrogenase from Corynebacterium glutamicum.

The NADP+ -dependent glutamate dehydrogenase from Corynebacterium glutamicum (CgGDH) is considered to be one of the key enzymes in the industrial fermentation of glutamate due to its high glutamate-producing activity. We determined the crystal structure of CgGDH complexed with NADP+ and 2-iminoglutarate. Among six subunits of hexameric CgGDH-binding NADP+ , only four subunits bind 2-iminoglutarate in a closed form, while the other two are in an open form. In the closed form, 2-iminoglutarate is bound to the substrate-binding site with the 2-imino group stacked by the nicotinamide ring of the coenzyme, suggesting a prehydride transfer state in a hypothesized reaction scheme with the imino intermediate. We also conducted MD simulations and provide insights into the extreme preference for the glutamate-producing reaction of CgGDH. DATABASE: The atomic coordinate and structure factors have been deposited in the RCSB PDB database under the accession number 5GUD.

Bioconversion of Iminodiacetonitrile to Iminodiacetic acid with whole cells of Lysinibacillus boronitolerans MTCC 107614 (IICT-akl252).

Biotechnological potential of nitrilases are prompting significant interest in finding the novel microbes capable of hydrolyzing nitriles. In this view, we have screened about 450 bacterial strains for nitrilase production using bioconversion of iminodiacetonitrile (IDAN) to iminodiacetic acid (IDA) through hydrolysis and obtained six nitrilase-producing isolates. Among these six isolates, IICT-akl252 was promising which was identified as Lysinibacillus boronitolerans. This is the first report on L. boronitolerans for nitrilase activity. Optimization of various medium and reaction parameters for maximizing the nitrilase production using whole cells in shake flask was carried out for L. boronitolerans IICT-akl252. Sucrose (2 %) as a carbon source attained better nitrilase yield while IDAN appeared to be the preferable inducer (0.2 %). The maximum IDA formation was achieved with 100 mM IDAN and 150 mg/ml cells at 30 °C and pH 6.5. After optimization of the culture and reaction conditions, the activity of nitrilase was increased by 2.3-fold from 27.2 to 64.5 U. The enzyme was stable up to 1 h at 50 °C. The enzyme was able to hydrolyze aliphatic, aromatic and heterocyclic nitrile substrates.

High-level production of Arthrobacter aurescens CYC705 nitrilase in Escherichia coli for biosynthesis of iminodiacetic acid.

Nitrilase from Arthrobacter aurescens CYC705 can hydrolyze the iminodiacetonitrile to iminodiacetic acid (IDA) efficiently, and its high-level production in Escherichia coli has not been established. In the present work, the production of this nitrilase expressed in E. coli BL21(DE3) with a recombinant plasmid pET28a-cyc705 was optimized. Various culture conditions and process parameters including medium components and concentrations, inducer types and concentrations, inducing temperature and time were systematically examined in a shake flask. After optimization, the OD600 , nitrilase activity, and productivity were obviously improved and achieved to 40.91 ± 1.341, 98.12 ± 1.248 U/mL, and 2,230 ± 28.36 U L(-1)  H(-1) , respectively, about 2.1-, 30-, and 33-fold increases as compared with those in the primary medium. Furthermore, four different fermentation strategies were adopted to scale up cultivation of the recombinant E. coli BL21(DE3)/pET28a-cyc705 in a 3.7-L fermenter. Substituting the peanut powder with fish peptone and accompanying with 1.0% glycerol feeding could significantly reduce the bubble production and shorten the fermentation time, which resulted in a nitrilase productivity of 4,653 ± 38.16 U L(-1) H(-1) that was about two times higher than that in a shake flask. The high-level production of A. aurescens CYC705 nitrilase established in this study will meet the need of industrial biosynthesis of IDA.

In situ generation of iminodiacetic acid groups on nanoporous alumina for the reversible immobilization of enzymes and other biomolecules.

Nanoporous alumina membranes were silanized with aminopropylsilane and iminodiacetic acid (IDA) groups were generated in situ by reaction with iodoacetate. The membranes were mounted in standard filter holders, connected to a HPLC system and saturated with selected metal ions. Cu(II) allowed the capture of chicken muscle lactate dehydrogenase with such stability, repeatability and reproducibility that Michaelis-Menten kinetics could be studied. The IDA surface was stable for months and could be depleted and regenerated with metal ions multiple times without appreciable loss of capacity. The binding of lactate dehydrogenase influenced the backpressure to the extent that could be expected for a monolayer according to Poiseuilles law.

Characterization of a nitrilase from Arthrobacter aurescens CYC705 for synthesis of iminodiacetic acid.

A nitrilase gene cyc705 from Arthrobacter aurescens CYC705 for synthesis of iminodiacetic acid (IDA) was cloned. This gene contained a 930 bp ORF, which encoded a polypeptide of 310 amino acids. A recombinant Escherichia coli BL21(DE3)/pET28a-cyc705 was constructed to achieve the heterologous expression of cyc705. This recombinant nitrilase was purified to homogeneity with a molecular weight of 36.7 kDa on SDS-PAGE and mass spectrometry, and characterized to be an oligomer of 14 subunits by gel permeation chromatography. Using iminodiacetonitrile (IDAN) as the substrate, the Vmax, Km, kcat and kcat/Km were 9.05 U mg(-1), 43.17 mM(-1), 94.1 min(-1) and 2.18×10(3) min(-1) M(-1), respectively. The optimum temperature and pH were 25°C and 5.8. The suitable substrates for the purified nitrilase were short-chain aliphatic dinitriles. High concentration of IDAN could be hydrolyzed to IDA in a shorter time.

Solvent effect investigation for the dioxovanadium (v) complexation with iminodiacetic acid on the basis of the Kamlet-Abboud-Taft equation.

Fits for the calculation of solvatochromic regression coefficients were done using the regression tool for the complexation of dioxovanadium(V) with iminodiacetic acid (IDA) and dissociation constants at T = 298 K and constant ionic strength of 0.1 mol dm-3 sodium perchlorate in different volume fractions of methanol (0 to 45 percent). A combination of potentiometric and UV spectrophotometric methods have been used for experimental measurements. Kamlet-Abboud-Taft (KAT) solvatochromic equation enables us to find out the contribution of various non specific and specific solute-solvent interactions. The results have been interpreted on the basis of the hydrogen-bond donor and acceptor ability and solvent polarity.

Crystal structures of vertebrate dihydropyrimidinase and complexes from Tetraodon nigroviridis with lysine carbamylation: metal and structural requirements for post-translational modification and function.

Lysine carbamylation, a post-translational modification, facilitates metal coordination for specific enzymatic activities. We have determined structures of the vertebrate dihydropyrimidinase from Tetraodon nigroviridis (TnDhp) in various states: the apoenzyme as well as two forms of the holoenzyme with one and two metals at the catalytic site. The essential active-site structural requirements have been identified for the possible existence of four metal-mediated stages of lysine carbamylation. Only one metal is sufficient for stabilizing lysine carbamylation; however, the post-translational lysine carbamylation facilitates additional metal coordination for the regulation of specific enzymatic activities through controlling the conformations of two dynamic loops, Ala(69)-Arg(74) and Met(158)-Met(165), located in the tunnel for the substrate entrance. The substrate/product tunnel is in the "open form" in the apo-TnDhp, in the "intermediate state" in the monometal TnDhp, and in the "closed form" in the dimetal TnDhp structure, respectively. Structural comparison also suggests that the C-terminal tail plays a role in the enzymatic function through interactions with the Ala(69)-Arg(74) dynamic loop. In addition, the structures of the dimetal TnDhp in complexes with hydantoin, N-carbamyl-ß-alanine, and N-carbamyl-ß-amino isobutyrate as well as apo-TnDhp in complex with a product analog, N-(2-acetamido)-iminodiacetic acid, have been determined. These structural results illustrate how a protein exploits unique lysines and the metal distribution to accomplish lysine carbamylation as well as subsequent enzymatic functions.

Screening and Improving the Recombinant Nitrilases and Application in Biotransformation of Iminodiacetonitrile to Iminodiacetic Acid.

In this study, several nitrilase genes from phylogenetically distinct organisms were expressed and purified in E. coli in order to study their ability to mediate the biotransformation of nitriles. We identified three nitrilases: Acidovorax facilis nitrilase (AcN); Alcaligenes fecalis nitrilase (AkN); and Rhodococcus rhodochrous nitrilase (RkN), which catalyzed iminodiacetonitrile (IDAN) to iminodiacetic acid (IDA). AcN demonstrated 8.8-fold higher activity for IDAN degradation as compared to AkN and RkN. Based on homology modeling and previously described 'hot spot' mutations, several AcN mutants were screened for improved activity. One mutant M3 (F168V/L201N/S192F) was identified, which demonstrates a 41% enhancement in the conversion as well as a 2.4-fold higher catalytic efficiency towards IDAN as compared to wild-type AcN.

In vivo visualization and quantification of (Disturbed) Oatp-mediated hepatic uptake and Mrp2-mediated biliary excretion of 99mTc-mebrofenin in mice.

UNLABELLED: Hepatic transport of (99m)Tc-mebrofenin through organic anion transport protein 1a and 1b (Oatp1a/1b) and multidrug resistance protein 2 (Mrp2) was investigated by small-animal SPECT. On the basis of the results, a noninvasive method to visualize and quantify disturbances in hepatic transport is proposed. METHODS: Friend virus B wild-type mice (untreated, bile duct-ligated, vehicle- or rifampicin-treated) and strain-matched knockout mice unable to express the uptake transporters Oatp1a/1b (Slco1a/1b(-/-)/(-/-)) or the efflux transporter Mrp2 (Abcc2(-/-)) were intravenously injected with (99m)Tc-mebrofenin (n = 3 per group). After dynamic small-animal SPECT and short CT acquisitions, time-activity curves of the liver and of the gallbladder and intestines were obtained and correlated with direct blood samples. RESULTS: Normal hepatobiliary clearance of (99m)Tc-mebrofenin was severely impaired in the bile duct-ligated animal, as evidenced by elevated hepatic tracer levels. In Slco1a/1b(-/-)/(-/-) mice, a lower area under the curve (AUC) for the liver (P = 0.014) was obtained and no activity was detected in the gallbladder and intestines. Renal rerouting was observed, along with an increase in the blood AUC (P = 0.01). Abcc2(-/-) mice had a higher liver AUC (P = 0.009), a delayed emergence time of (99m)Tc-mebrofenin in the gallbladder (P = 0.009), and a lower AUC for the gallbladder and intestines (P = 0.001). The blood curve was similar to that of wild-type mice. (99m)Tc-mebrofenin disposition was altered after rifampicin treatments. We observed a dose-dependent delayed time point at which tracer maximized in liver, an increased AUC for liver, and a lower AUC for gallbladder and intestines (P = 0.042, 0.034, and 0.001, respectively, highest dose). Emergence in the gallbladder occurred later (P = 0.009, highest dose), and blood AUC was higher (P = 0.006). CONCLUSION: The current study visualized and quantified hepatic uptake and biliary efflux of (99m)Tc-mebrofenin. Our results demonstrated the possibility of discriminating, on a quantitative level, between lack of functional activity of sinusoidal uptake versus that of biliary efflux transporters.

Biosynthesis of iminodiacetic acid from iminodiacetonitrile by immobilized recombinant Escherichia coli harboring nitrilase.

Iminodiacetic acid (IDA) is widely used as an intermediate in the manufacture of chelating agents, glyphosate herbicides and surfactants. In the current work, the fragment with the length of 1,110 bp encoding the Acidovorax facilis nitrilase was obtained. The recombinant nitrilase expressed in Escherichia coli BL21 (DE3) was successfully used in the production of IDA from iminodiacetonitrile. To improve the stability of operation, the recombinant cells were entrapped in polyvinyl alcohol (PVA) and sodium alginate (SA) copolymer. The maximum relative nitrilase activity with 98.1% was further observed at 1.0% SA, 8.0% PVA, 1.0% CaCl(2), and 5.0% wet cells, under conditions of 1.0% iminodiacetonitrile in distilled water and a temperature of 40°C, respectively. The entrapped cells facilitated easy separation and good recycling compared with free cells. Moreover, the immobilized cells showed good operation and storage stability. This report is the first to describe IDA preparation using immobilized recombinant E. coli harboring nitrilase.

Novel multifunctional chitosan-GMA-IDA-Cu(II) nanospheres for high dynamic range characterization of the human plasma proteome.

In this study, we describe characterization of the human plasma proteome based on analysis with multifunctional chitosan-GMA-IDA-Cu(II) nanospheres. Chitosan-GMA-IDA-Cu(II) nanospheres with diameters of 20 to 100 nm have unique properties due to multifunctional chemical moieties, high surface area, high capacity, good dispersibility in buffer solution as well as good biocompatibility and chemical stability which improves their specific interaction with peptides and proteins of the human plasma using different binding buffers. Combining these chitosan-GMA-IDA-Cu(II) nanospheres with MS spectrometry results in a novel strategy which should make it possible to characterize the plasma proteome in a single test. Peptides and proteins adsorbed on the nanosphere can be directly detected by MALDI-TOF-MS. The eluted lower molecular weight peptides and proteins are identified by nano-LC-ESI-MS/MS. A total of 842 unique LMW peptides and 1,682 human unredundant proteins IDs were identified in two different binding buffers, which included relatively low-level proteins (e.g., pg/mL of IL3 Interleukin-3) co-distributed with high-abundance proteins (e.g., 35-55 mg/mL level serum albumin). As such, this nanosphere technique selectively enabled the identification of proteins over a dynamic range of greater than 8 orders of magnitude. Considering this capacity for selective enrichment of peptides and proteins in human plasma, and the large number of LMW peptides and proteins which can be identified, this method promises to accelerate discovery of biomarkers for clinical application.

Metal ion binding patterns of acyclovir: molecular recognition between this antiviral agent and copper(II) chelates with iminodiacetate or glycylglycinate.

In order to deepen on metal-binding patterns of acyclovir (acv), {[Cu(IDA)(acv)]·2MeOH}(n) (1) and [Cu(glygly)(acv)]·H(2)O (2) compounds have been synthesized and investigated by X-ray crystallography as well as spectral and thermal methods. These compounds have been chosen upon the assumption that iminodiacetate (IDA) and glycylglycinate (glygly) chelating ligands would bind copper(II) with mer-tridentate conformation, supplying two terminal H-acceptor carboxylate groups (IDA) or one H-acceptor carboxylate and one H-donor primary amino group (glygly). The main aim of this work was to clarify if the amino group of glygly can build an intra-molecular interligand H-bonding interaction to reinforce the Cu-N7(acv) bond. Our results are discussed in the context of an up-to-date critical look regarding the related structural information. From the viewpoint of molecular recognition, the structure of 1 shows that the chelate-nucleoside recognition only involves the Cu-N7(acv) coordination bond. In contrast, the molecular complex of 2 exhibits the Cu-N7(acv) coordination bond reinforced by an intra-molecular (glygly)N-H···O6(acv) interaction (2.961(3)Å, 140.5°).

Preparation and characterization of iminodiacetic acid-functionalized magnetic nanoparticles and its selective removal of bovine hemoglobin.

In this study, a novel route for the preparation of magnetite (Fe(3)O(4)) nanoparticles (NPs) with immobilized metal affinity ligand iminodiacetic acid (IDA) charged with Cu(2+) was developed. First, magnetite nanoparticles were synthesized by a hydrothermal method. Charged with Cu(2+), the magnetic nanoparticles (MNPs) were applied to separate a model protein mixture of bovine hemoglobin (BHb) and bovine serum albumin (BSA). They could be separated completely and showed low non-specific adsorption. The morphology, structure and composition of the magnetite MNPs were characterized by transmission electron microscopy, power x-ray diffraction, x-ray photoelectron spectrometry and Fourier transform infrared spectroscopy. The resulting magnetite MNPs charged with Cu(2+) show not only a strong magnetic response to externally applied magnetic field, but are also highly specific to protein BHb. It is interesting that MNPs modified with metal ligands showed a property of magnetic colloid photonic crystals. Furthermore, they could efficiently remove the abundant protein bovine hemoglobin from bovine blood. They have potential application in removing abundant protein in proteomic analysis.

Renal imino acid and glycine transport system ontogeny and involvement in developmental iminoglycinuria.

Renal maturation occurs post-natally in many species and reabsorption capacity at birth can vary substantially from the mature kidney. However, little is known regarding the maturation of amino acid transport mechanisms, despite the well-known physiological state of developmental iminoglycinuria. Commonly seen during early infancy, developmental iminoglycinuria is a transient version of the persistent inherited form of the disorder, referred to as iminoglycinuria, and manifests as a urinary hyperexcretion of proline, hydroxyproline and glycine. The transporters involved in developmental iminoglycinuria and their involvement in the improvement of renal reabsorption capacity remain unknown. qPCR (quantitative real-time PCR) and Western blot analysis in developing mouse kidney revealed that the expression of Slc6a18, Slc6a19, Slc6a20a and Slc36a2 was lower at birth (approx. 3.4-, 5.0-, 2.4- and 3.0-fold less than adult kidney by qPCR respectively) and increased during development. Furthermore, immunofluorescence confocal microscopy demonstrated the absence of apical expression of Slc6a18, Slc6a19, Slc6a20a and the auxiliary protein collectrin in kidneys of mice at birth. This correlated with the detection of iminoglycinuria during the first week of life. Iminoglycinuria subsided (proline reduction preceded glycine) in the second week of life, which correlated with an increase in the expression of Slc6a19 and Slc6a20a. Mice achieved an adult imino acid and glycine excretion profile by the fourth week, at which time the expression level of all transporters was comparable with adult mice. In conclusion, these results demonstrate the delayed expression and maturation of Slc6a18, Slc6a19, Slc6a20a and Slc36a2 in neonatal mice and thus the molecular mechanism of developmental iminoglycinuria.

Sodium translocation by the iminoglycinuria associated imino transporter (SLC6A20).

The system IMINO transporter plays an essential role in the transport of proline and hydroxyproline in the intestine and kidney. Its molecular correlate has been identified and named SIT1 or IMINO (SLC6A20). Initial characterization of the transporter showed it to be Na(+) and Cl(-)-dependent, but the stoichiometry remained unresolved. Using homology modeling along the structure of the bacterial leucine transporter LeuT, we identified two highly conserved Na(+)-binding sites and a putative Cl(-)-binding site. Mutation of all residues in the two proposed Na(+)-binding sites revealed that most of them were essential for uptake and completely inactivated the transporter. However, mutants A22V (Na(+)-binding site 1) and mutants S20A, S20G, S20G/G405S (Na(+)-binding site 2) were partially active and characterized further. Flux studies suggested that mutations of Na(+)-binding site 1 caused a decrease of the Na(+)-K(0.5), whereas mutations of site 2 increased the K(0.5). Mutation of Na(+)-binding site 1 also changed the ion selectivity of the IMINO transporter. IMINO actively translocates (36)Cl(-) demonstrating that the proposed chloride binding site is used in the transporter. Accumulation experiments and flux measurements at different holding potentials showed that the transporter can work as a 2Na(+)/1Cl(-)-proline cotransporter. The proposed homology model allows to study mutations in IMINO associated with iminoglycinuria.

Directed formation of lipid membrane microdomains as high affinity sites for His-tagged proteins.

Lipid membranes composed of an iminodiacetic acid functionalized lipid, DSIDA, in a POPC matrix exhibited switchable properties via Cu(2+) recognition to rapidly assemble microdomains that act as high affinity sites for His-tagged proteins. The microdomains demonstrated an order of magnitude enhanced affinity for the proteins compared to homogeneously functionalized POPC membranes with Ni(2+)-NTA DOGS or Cu(2+)-DOIDA, while a rapid release and restoration of the original membrane was accomplished with micromolar concentrations of EDTA.