Stereoselective histidine sensor based on molecularly imprinted sol-gel films

A piezoelectric sensor coated with a thin molecularly imprinted sol-gel film has been developed for the determination of L-histidine in the liquid phase. Without preprotection, L-histidine was imprinted directly into silica sol-gel films that consisted of a hybrid mixture of functionalized organosilicon precursors (phenyltrimethoxysilane and methyltrimethoxysolane). The viscoelasticity of the film in the air and in buffer solution has been studied by the piezoelectric quartz crystal impedance technique. The binding of L-histidine to the imprinted film in the liquid phase was investigated by the piezoelectric microgravimetry and electrochemical impedance technique. Scatchard analysis showed that the maximum binding site (Qmax) of the L-histidine imprinted sol-gel film is about 23.7 micromol/g. A linear range from 5.0x10(-8) to 1.0x10(-4) M for a detection of L-histidine has been observed with a detection limit of 2.5x10(-8) M for S/N=3. The proposed imprinted sol-gel sensor exhibits good stability, high specificity, and excellent stereoselectivity.     

Variability in ionization state,stoichiometry and aggregation in histidine complexes with formic acid

The crystal structures of the complexes of L and DL histidine with formic acid have been determined as part of an effort to define biologically and evolutionarily important interactions and aggregation patterns. In terms of ionization state and stoichiometry they may be described as L-histidine formate formic acid and DL-histidine formate monohydrate respectively. In the L-histidine complex, amino acid molecules arranged in head-to-tail sequences centred around 2₁ screw axes are interconnected by formic acid molecules and formate ions. Histidine-formate interactions in the structure gives rise to a characteristic interaction pattern involving a linear array of alternating imidazole groups and formate ions. In DL-histidine formale monohydrate, head-to-tail sequences involving glide related molecules are interconnected through main chain-side chain interactions leading to amino acid layers. The layers are held together by formate ions and water molecules arranged in strings along which the ion and the molecule alternate. The patterns of amino acid aggregation in histidine complexes exhibit considerably higher variability than those in complexes involving arginine and lysine do. X-ray studies on crystalline complexes involving amino and peptides Part XXIX.     

Histidine production by a regulatory mutant of Streptomyces coelicolor

Streptomyces coelicolor mutant RF-59, isolated as a revertant of a histidine auxotroph after mutagenic treatment with N-methylN'-nitro-N-nitrosoguanidine, was found to accumulate L-histidine. The mutant was sensitive to 2-thiazo-lealanine and L-2,4-diaminobutyric acid and partially sensitive to alpha-methylhistidine but resistant to 1,2,4-triazolealanine, indicating that repression of the histidine operon was modified in the mutant. Culture conditions were investigated, and optimal media for L-histidine production were developed, resulting in L-histidine accumulation of 2.1 to 3.5 g/liter.     

Histidine production by a regulatory mutant of Streptomyces coelicolor.

Streptomyces coelicolor mutant RF-59, isolated as a revertant of a histidine auxotroph after mutagenic treatment with N-methylN'-nitro-N-nitrosoguanidine, was found to accumulate L-histidine. The mutant was sensitive to 2-thiazo-lealanine and L-2,4-diaminobutyric acid and partially sensitive to alpha-methylhistidine but resistant to 1,2,4-triazolealanine, indicating that repression of the histidine operon was modified in the mutant. Culture conditions were investigated, and optimal media for L-histidine production were developed, resulting in L-histidine accumulation of 2.1 to 3.5 g/liter.     

DNA Single Strand Breakage by H2O2 and Ferric or Cupric Ions: Its Modulation by Histidine

The role of histidine on DNA breakage induced by hydrogen peroxide (H₂O₂) and ferric ions or by H₂O₂ and cupric ions was studied on purified DNA. L-histidine slightly reduced DNA breakage by H₂O₂ and Fe³⁺ but greatly inhibited DNA breakage by H₂O₂ and Cu²⁺. However, only when histidine was present, the addition of EDTA to H₂O₂ and Fe³⁺ exhibited a bimodal dose response curve depending on the chelator metal ratio. The enhancing effect of histidine on the rate of DNA degradation by H₂O₂ was maximal at a chelator metal ratio between 0.2 and 0.5, and was specific for iron. When D-histidine replaced L-histidine, the same pattern of EDTA dose response curve was observed. Superoxide dismutase greatly inhibited the rate of DNA degradation induced by H₂O₂, Fe³⁺, EDTA and L-histidine involving the superoxide radical.

These studies suggest that the enhancing effect of histidine on the rate of DNA degradation by H₂O₂ and Fe³⁺ is mediated by an oxidant which could be a ferrous-dioxygen-ferric chelate complex or a chelate-ferryl ion.     

Crystal structure of activated HutP; an RNA binding protein that regulates transcription of the hut operon in Bacillus subtilis

HutP is an L-histidine-activated RNA binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis by binding to cis-acting regulatory sequences on the hut mRNA. The crystal structure of HutP complexed with an L-histidine analog showed a novel fold; there are four antiparallel beta strands in the central region of each monomer, with two alpha helices each on the front and back. Two HutP monomers form a dimer, and three dimers are arranged in crystallographic 3-fold symmetry to form a hexamer. A histidine analog was located in between the two monomers of HutP, with the imidazole group of L-histidine hydrogen bonded to Glu81. An activation mechanism is proposed based on the identification of key residues of HutP. The HutP binding region in hut mRNA was defined: it consists of three UAG trinucleotide motifs separated by four spacer nucleotides. Residues of HutP potentially important for RNA binding were identified.     

Studies on the production and assessment of experimental histidinemia in the rat.

Intraperitoneal administration to rats of D- or DL-alpha-hydrazinoimidazolylpropionic acid was found to produce a substantial inactivation of hepatic histidine ammonia-lyase (EC 4.3.1.3) in vivo. Proportional to this loss in enzyme activity was an impairment of the ability of treated rats to oxidize L-[ring-2-14C] histidine to 14CO2. Rats in which hepatic histidine ammonia-lyase activity was either depressed by DL-hydrazinoimidazolylproprionic acid injection or elevated by feeding a high protein diet displayed proportionately altered rates of 3H2O release into plasma water following L-[3-3H] histidine administration. Plasma L-histidine clearance following loading with this amino acid was similarly affected by these treatments. Administration of DL-alphal-hydrazinoimisazolylproprionic acid to rats was also found to inactivate non-specifically pyridoxal 5-phosphate enzymes in vivo; pyridoxine injection was found to reverse the DL-alpha-hydrazinoimidazolylproprionic acid-induced inactivation of hepatic aspartate aminotransferase (EC 2.6.1.1) in vivo, but not that of hepatic histidine ammonia-lyase. These findings demonstrate that histidine ammonia-lyase is the rate-limiting factor in L-histidine degradation in the rat. The potential usefulness of DL-hydrazinoimidazolylproprionic acid in the production of an animal model for histidinemia (hereditary histidine ammonia-lyase deficiency) is discussed.     

Reversible stepwise mechanism involving a carbanion intermediate in the elimination of ammonia from L-histidine catalyzed by histidine ammonia-lyase.

L-Histidine labeled with deuterium at the C-5' position of the imidazole ring, L-[5'-2H]histidine (His-5'-D), was used as a probe for investigating a stepwise reversible mechanism via a carbanion intermediate in the elimination of ammonia catalyzed by histidine ammonia-lyase (EC 4.3.1.3). The labeled L-histidine (His-5'-D) (2.45 mM) was incubated with histidine ammonia-lyase (200 units) from Pseudomonas fluorescens at pH 7.0 or 9.0 at 25.0 degrees C for 24 h. The time course of the reaction was examined to determine the rates of enzyme-catalyzed hydrogen exchange at C-5' of L-histidine and urocanic acid. The finding of the enzyme-catalyzed hydrogen exchange at C-5' of both L-histidine and urocanic acid in the presence of L-histidine provided a rational explanation for a stepwise reversible mechanism via a carbanion intermediate in the elimination reaction. The rate of increase in the concentration of urocanic acid exchanged with hydrogen (UA-5'-H) did not depend on the formation rate of urocanic acid and UA-5'-H was continuously formed at a constant rate (25.6 microM/h) even after the completion of urocanic acid formation. These observations suggested the presence of the reversible reaction of urocanic acid and a carbanion intermediate. Since there was only a minor contribution for the formation of UA-5'-H from L-histidine exchanged with solvent hydrogen (His-5'-H), the main pathway in the enzymatic reaction of His-5'-D must be the formation of UA-5'-D via a carbanion intermediate (carbanion-D). Regeneration of the carbanion-D from UA-5'-D by its reverse reaction and subsequent hydrogen incorporation at C-5' would contribute to a large extent for the formation of UA-5'-H. The stability of carbanion was also demonstrated to be approximately three times higher at pH 7.0 than at pH 9.0.     

Histidine ammonia-lyase from rat liver. Purification, properties, and inhibition by substrate analogues.

Histidine ammonia-lyase (EC 4.3.1.3) from rat liver was purified more than 250-fold to near homogeneity. Electrophoretic determinations indicated a native molecular weight of approximately 200,000. The enzyme has a pH optimum of approximately pH 8.5. The minimum Km for L-histidine was 0.5 mM at pH 9.0. The Michaelis constant in the physiological pH range was, however, more than 2.0 mM. D-alpha-hydrazinoimidazolylpropionic acid was found to be a potent competitive inhibitor of liver histidine ammonia-lyase (Kis=75 muM); the L enantiomer of this compound was less effective in this regard. The enzyme was also inhibited competitively by L-histidine hydroxamate (Kis=0.4 mM), and to a lesser extent by L-histidinol, D-histidine, and glycine. Failure of a wide variety of other histidine analogues to inhibit the enzyme substantially indicates high specificity of the active site for L-histidine. No alternate substrates were identified for the enzyme. DL-alpha-Hydrazinophenylpropionic acid, the alpha-hydrzino analogue of phenylalanine, was similarly shown to be a very potent competitive inhibitor of a mechanistically similar L-phenylalanine ammonia-lyase purified from Rhodotorula glutinis. The properties of histidine ammonia-lyase from rat liver differ significantly from those of the enzyme from Pseudomonas fluorescens which has been studied most extensively to date.     

L-Histidine production by histidase-less regulatory mutants of Serratia marcescens constructed by transduction.

2-Methylhistidine (2MH) and 1,2,4-triazole-3-alanine (TRA) inhibited the growth of Serratia marcescens. These inhibitory effects were counteracted by L-histidine. Enzymatic studies showed that 2MH acts as a false feedback inhibitor and TRA acts as both a false feedback inhibitor and a repressor. Mutants resistant to each analog were isolated from a histidase-less mutant, because the wild-type strain possesses a potent histidase activity. 2MH-resistant mutants had a feedback-insensitive phosphoribosyltransferase, but they produced only small amounts of L-histidine. TRA-resistant mutants were divided into two types according to their histidine productivity. A mutant of one type produced about 8 mg of L-histidine per ml and had about a 10-fold increase in the enzyme levels of histidine biosynthesis. Moreover, this mutant had a partially feedback-insensitive phosphoribosyltransferase. A mutant of the second type produced only a small amount of L-histidine and had only derepressed enzyme levels. Accordingly, strains possessing the genetic alterations in both 2MH- and TRA-resistant mutants were constructed by PS20-mediated transduction. They had both feedback-insensitive phosphoribosyltransferase and derepressed enzyme levels. The representative strain HT-2604 produced about 17 mg of L-histidine per ml.     

L-Histidine production by histidase-less regulatory mutants of Serratia marcescens constructed by transduction.

2-Methylhistidine (2MH) and 1,2,4-triazole-3-alanine (TRA) inhibited the growth of Serratia marcescens. These inhibitory effects were counteracted by L-histidine. Enzymatic studies showed that 2MH acts as a false feedback inhibitor and TRA acts as both a false feedback inhibitor and a repressor. Mutants resistant to each analog were isolated from a histidase-less mutant, because the wild-type strain possesses a potent histidase activity. 2MH-resistant mutants had a feedback-insensitive phosphoribosyltransferase, but they produced only small amounts of L-histidine. TRA-resistant mutants were divided into two types according to their histidine productivity. A mutant of one type produced about 8 mg of L-histidine per ml and had about a 10-fold increase in the enzyme levels of histidine biosynthesis. Moreover, this mutant had a partially feedback-insensitive phosphoribosyltransferase. A mutant of the second type produced only a small amount of L-histidine and had only derepressed enzyme levels. Accordingly, strains possessing the genetic alterations in both 2MH- and TRA-resistant mutants were constructed by PS20-mediated transduction. They had both feedback-insensitive phosphoribosyltransferase and derepressed enzyme levels. The representative strain HT-2604 produced about 17 mg of L-histidine per ml.     

Protection of transfective poliovirion RNA by histidine

Transfectivity titers of RNA preparations obtained from purified poliovirions in phosphate-buffered saline using phenol were low. Addition of tissue culture nutrient medium to the virions prior to extractin with phenol increased the RNA titers 100-1000-fold. The 32 solute differences between the phosphate-buffered saline and the nutrient medium were divided into three blocs for testing. Only the bloc containing the 13 amino acids of the nutrient medium enabled the preparation of high-titer RNA. Tests of the individuals amino acids revealed that L-histidine has high activity, L-cystine and L-glutamine moderate activity, and the remaining ten zero or very slight activity. Five congeners of L-histidine, viz. D-histidine, histamine, L-histidine methyl ester, alpha-N-acetyl-L-histidine, and L-histidyl-glycine, also had high activity; but imidazole had no activity. The histidine effect was obtained whether transfection was enhanced by DEAE-dextran or by bentonite. Histidine was fully effective only when it was added to the virions before or very shortly after the phenol; later additions of histidine were progressively less effective. Without added histidine, RNA preparations made very rapidly and inoculated promptly showed high transfectivity titers, but the transfectivity was highly labile; with histidine present, the high RNA titers were stable. Histidine did not reactivate the inactivated RNA.     

pH-dependence of 13C chemical shifts and 13C,H coupling constants in imidazole and L-histidine.

The pH-dependence of selected 13C chemical shifts reflects the state of ionization of the imidazole ring in both imidazole and L-histidine. Titration of the amino and carboxyl groups of histidine also perturbs the shifts. The coupling constants 1J (13C(2),H) and 1J (13C(5),H) for both compounds also vary with pH, but in L-histidine these constants are relatively insensitive to the titration of groups outside the imidazole ring.     

操纵子前导区的修饰及PRPP合成途径的加强对大肠杆菌积累L-组氨酸的影响

目的:基于转酮酶基因缺失菌株MG1655-ΔtktA,研究启动子替换L-组氨酸操纵子前导区及6-磷酸葡萄糖脱氢酶基因zwf、6-磷酸葡萄糖酸脱氢酶基因gnd、PRPP合成酶基因prs的过表达对大肠杆菌产L-组氨酸的影响。方法:通过Red重组系统用T5启动子替换L-组氨酸操纵子前导区;构建gnd和zwf串联表达载体gnd-zwf-pSTV28,prs表达载体prs-pQE30。通过摇瓶发酵,考察上述改造对大肠杆菌积累L-组氨酸的影响。结果:测定结果显示,改造菌株的发酵液中均能实现L-组氨酸积累,平均分别为MG1655-ΔtktA-PT5,60.12 mg/L;MG1655-ΔtktA-PT5(prs-pQE30),66.47mg/L;MG1655-ΔtktA-PT5(zwf-gnd-pSTV28),89.69 mg/L;MG1655-ΔtktA-PT5(prs-pQE30,zwf-gnd-pSTV28),111.56 mg/L。结论:L-组氨酸操纵子前导区的修饰使菌株合成L-组氨酸的能力大大增强,而氧化戊糖磷酸途径的加强和PRPP合成酶活性的提高能够进一步提高产量。     

Crystallization and preliminary X-ray diffraction studies of the metal-ion-mediated ternary complex of the HutP protein with L-histidine and its cognate RNA

HutP is an RNA-binding protein that regulates the expression of the Bacillus subtilis hut operon by binding to cis-acting regulatory sequences within hut mRNA, exclusively in the presence of L-histidine. We recently solved the crystal structure of a binary complex (HutP with an L-histidine analog) that revealed a novel RNA-binding fold, and identified the important residues that interact with the L-histidine analog. In addition, we have defined the minimal RNA binding segment that is required for HutP recognition. Interestingly, we showed that ternary complex formation depends on the availability of not only L-histidine but also divalent metal ions. Here we report the crystallization and preliminary X-ray diffraction analysis of the HutP ternary complex. The ternary complex was crystallized in the presence of Mg2+ along with L-histidine and hut mRNA, using the hanging drop vapor diffusion method. The crystal belongs to the R3 space group, with unit cell parameters a=b=75.30 A, c=133.8 A. A complete data set at 1.60 A was collected.     

Crystalline L-histidine ammonia-lyase of Achromobacter liquidum. Crystallization and enzymic properties.

Crystalline L-histidine ammonia-lyase of Achromobacter liquidum was prepared with a 24% recovery of the activity. The specific activity of the pure enzyme (63 mumol of urocanic acid min-1 mg-1) is similar to those so far reported for the enzyme from other sources. The purified enzyme appeared to be homogeneous by analytical disc electrophoresis and isoelectric focusing (pI = 4.95). The molecular weight determined by Sephadex G-200 gel filtration is 200000. The optimum pH is 8.2, and the optimum temperature is 50 degrees C. The enzyme showed strict specificity to L-histidine (Km = 3.6 mM). Several histidine derivatives are not susceptible to the enzyme but do inhibit the enzyme activity competitively; the most effective inhibitors are L-histidine methyl ester (Ki = 3.66 mM) and beta-imidazole lactic acid (Ki = 3.84 mM). L-Histidine hydrazide (Ki = 36 mM) and imidazole (Ki = 6 mM) noncompetitively inhibited the enzyme EDTA markedly inhibited enzyme activity and this inhibition were reversed by divalent metal ions such as Mn2+, Co2+ Zn2+, Ni2+, Mg2+, and Ca2+. These results suggest that the presence of divalent metal ions is necessary for the catalytic activity of histidine ammonia-lyase. Sodium borohydride and hydrogen peroxide inhibited the enzyme activity.     

Kinetics and thermodynamics of annexin A1 binding to solid-supported membranes: a QCM study

By means of the quartz crystal microbalance (QCM) technique, the interaction of annexin A1 with lipid membranes was quantified using solid-supported bilayers immobilized on gold electrodes deposited on 5 MHz quartz plates. Solid-supported lipid bilayers were composed of a first octanethiol monolayer chemisorbed on gold and a physisorbed phospholipid monolayer obtained from vesicle fusion. This experimental setup enabled us to determine for the first time rate constants and affinity constants of annexin A1 binding to phosphatidylserine-containing layers as a function of the calcium ion concentration in solution and the cholesterol content within the outer leaflet of the solid-supported bilayer. The results reveal that a decrease in Ca(2+) concentration from 1 mM to 100 microM significantly increases the rate of annexin A1 binding to the membrane independent of the cholesterol content. However, the presence of cholesterol in the membrane altered the affinity constants considerably. While the association constant decreases with decreasing Ca(2+) concentration in the case of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) membranes lacking cholesterol, it remains high in the presence of cholesterol.     

紫外线与亚硝酸钠复合诱变选育L-组氨酸产生菌

以1株谷氨酸棒杆菌(Corynebacterium glutamicum)S_6作为出发菌株,利用亚硝酸钠(NaNO_2)、紫外线(UV)进行诱变,通过实验证明亚硝酸钠诱变时间在180 s后致死率达到80%,紫外线在照射30 s后致死率达到80%。诱变后的突变菌株经6-巯基嘌呤结构类似物的抗性平板筛选,最终筛得3株菌,Y_1产L-组氨酸量达到331 mg/L,比出发菌株S_6高了5.08%,Z_2产L-组氨酸量达到325 mg/L,比出发菌株S_6高了1.9%,F_6产L-组氨酸量达到330 mg/L,比出发菌株S_6高了7.14%。结果显示,经紫外线与亚硝酸钠复合诱变后的菌株F_6产L-组氨酸的产量最高,比亚硝酸钠诱变后的菌株产L-组氨酸量提高2.06%,比紫外线诱变后的菌株产L-组氨酸量提高5.24%。