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Feedback control is a universal feature of cell signaling pathways. Naked/NKD is a widely conserved feedback regulator of Wnt signaling which controls animal development and tissue homeostasis. Naked/NKD destabilizes Dishevelled, which assembles Wnt signalosomes to inhibit the β-catenin destruction complex via recruitment of Axin. Here, we discover that the molecular mechanism underlying Naked/NKD function relies on its assembly into ultra-stable decameric core aggregates via its conserved C-terminal histidine cluster (HisC). HisC aggregation is facilitated by Dishevelled and depends on accumulation of Naked/NKD during prolonged Wnt stimulation. Naked/NKD HisC cores co-aggregate with a conserved histidine cluster within Axin, to destabilize it along with Dishevelled, possibly via the autophagy receptor p62, which binds to HisC aggregates. Consistent with this, attenuated Wnt responses are observed in CRISPR-engineered flies and human epithelial cells whose Naked/NKD HisC has been deleted. Thus, HisC aggregation by Naked/NKD provides context-dependent feedback control of prolonged Wnt responses., Competing Interests: MG, MR, JF, JM, MB No competing interests declared, (© 2020, Gammons et al.)

L-histidine (HIS) is an essential amino acid with unique roles in proton buffering, metal ion chelation, scavenging of reactive oxygen and nitrogen species, erythropoiesis, and the histaminergic system. Several HIS-rich proteins (e.g., haemoproteins, HIS-rich glycoproteins, histatins, HIS-rich calcium-binding protein, and filaggrin), HIS-containing dipeptides (particularly carnosine), and methyl- and sulphur-containing derivatives of HIS (3-methylhistidine, 1-methylhistidine, and ergothioneine) have specific functions. The unique chemical properties and physiological functions are the basis of the theoretical rationale to suggest HIS supplementation in a wide range of conditions. Several decades of experience have confirmed the effectiveness of HIS as a component of solutions used for organ preservation and myocardial protection in cardiac surgery. Further studies are needed to elucidate the effects of HIS supplementation on neurological disorders, atopic dermatitis, metabolic syndrome, diabetes, uraemic anaemia, ulcers, inflammatory bowel diseases, malignancies, and muscle performance during strenuous exercise. Signs of toxicity, mutagenic activity, and allergic reactions or peptic ulcers have not been reported, although HIS is a histamine precursor. Of concern should be findings of hepatic enlargement and increases in ammonia and glutamine and of decrease in branched-chain amino acids (valine, leucine, and isoleucine) in blood plasma indicating that HIS supplementation is inappropriate in patients with liver disease.

Amyloid-β peptide (Aβ) plays a central role in Alzheimer's disease (AD) pathogenesis. Besides extracellular Aβ, intraneuronal Aβ (iAβ) has been suggested to contribute to AD onset and development. Based on reported in vitro Aβ-DNA interactions and nuclear localization of iAβ, the interference of iAβ with the normal DNA expression has recently been proposed as a plausible pathway by which Aβ can exert neurotoxicity. Employing the sedimentation assay, thioflavin T fluorescence, and dynamic light scattering we have studied effects of zinc ions on binding of RNA and single- and double-stranded DNA molecules to Aβ42 aggregates. It has been found that zinc ions significantly enhance the binding of RNA and DNA molecules to pre-formed β-sheet rich Aβ42 aggregates. Another type of Aβ42 aggregates, the zinc-induced amorphous aggregates, was demonstrated to also bind all types of nucleic acids tested. To evaluate the role of the Aβ metal-binding domain's histidine residues in Aβ-nucleic acid interactions mediated by zinc, Aβ16 mutants with substitutions H6R and H6A-H13A and rat Aβ16 lacking histidine residue 13 were used. The zinc-induced interaction of Aβ16 with DNA was shown to critically depend on histidine residues 6 and 13. However, the inclusion of H6R mutation in Aβ42 peptide did not affect DNA binding to Aβ42 aggregates. Since oxidative and/or nitrosative stresses implicated in AD pathogenesis are known to release zinc ions from metallothioneins in cytoplasm and cell nuclei, our findings suggest that intracellular zinc can be an important player in iAβ-nucleic acid interactions.

Trypanosoma cruzi, the aetiological agent of Chagas's disease, metabolizes glucose, and after its exhaustion, degrades amino acids as energy source. Here, we investigate histidine uptake and its participation in energy metabolism. No putative genes for the histidine biosynthetic pathway have been identified in genome databases of T. cruzi, suggesting that its uptake from extracellular medium is a requirement for the viability of the parasite. From this assumption, we characterized the uptake of histidine in T. cruzi, showing that this amino acid is incorporated through a single and saturable active system. We also show that histidine can be completely oxidised to CO2. This finding, together with the fact that genes encoding the putative enzymes for the histidine - glutamate degradation pathway were annotated, led us to infer its participation in the energy metabolism of the parasite. Here, we show that His is capable of restoring cell viability after long-term starvation. We confirm that as an energy source, His provides electrons to the electron transport chain, maintaining mitochondrial inner membrane potential and O2 consumption in a very efficient manner. Additionally, ATP biosynthesis from oxidative phosphorylation was found when His was the only oxidisable metabolite present, showing that this amino acid is involved in bioenergetics and parasite persistence within its invertebrate host.

Hepatic encephalopathy (HE) is related to variations in the nitric oxide (NO) synthesis and oxidative/nitrosative stress (ONS), and asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthases (NOSs). In the present study we compared the effects of acute liver failure (ALF) in the rat TAA model on ADMA concentration in plasma and cerebral cortex, and on the activity and expression of the ADMA degrading enzyme, dimethylarginine dimethylaminohydrolase (DDAH), in brain and liver. ALF increased blood and brain ADMA, and the increase was correlated with decreased DDAH activity in both brain and liver. An i.p. administration of histidine (His), an amino acid reported to alleviate oxidative stress associated with HE (100 mg/kg b.w.), reversed the increase of brain ADMA, which was accompanied by the recovery of brain DDAH activity (determined ex vivo), and with an increase of the total NOS activity. His also activated DDAH ex vivo in brain homogenates derived from control and TAA rats. ALF in this model was also accompanied by increases of blood cyclooxygenase activity and blood and brain TNF-α content, markers of the inflammatory response in the periphery, but these changes were not affected by His, except for the reduction of TNF-α mRNA transcript in the brain. His increased the total antioxidant capacity of the brain cortex, but not of the blood, further documenting its direct neuroprotective power., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In all organisms, several distinct stand-alone pseudouridine synthase (PUS) family enzymes are expressed to isomerize uridine into pseudouridine (Ψ) by specific recognition of RNAs. In addition, Ψs are generated in Archaea and Eukaryotes by PUS enzymes which are organized as ribonucleoprotein particles (RNP)--the box H/ACA s/snoRNPs. For this modification system, a unique TruB-like catalytic PUS subunit is associated with various RNA guides which specifically target and secure substrate RNAs by base-pairing. The archaeal Cbf5 PUS displays the special feature of exhibiting both RNA guide-dependent and -independent activities. Structures of substrate-bound TruB and H/ACA sRNP revealed the importance of histidines in positioning the target uridine in the active site. To analyze the respective role of H60 and H77, we have generated variants carrying alanine substitutions at these positions. The impact of the mutations was analyzed for unguided modifications U(55) in tRNA and U2603 in 23S rRNA, and for activity of the box H/ACA Pab91 sRNP enzyme. H77 (H43 in TruB), but not H60, appeared to be crucial for the RNA guide-independent activity. In contrast to earlier suggestions, H60 was found to be noncritical for the activity of the H/ACA sRNP, but contributes together with H77 to the full activity of H/ACA sRNPs. The data suggest that a similar catalytic process was conserved in the two divergent pseudouridylation systems., (© 2015 Tillault et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Super secondary structures of proteins have been systematically searched and classified, but not enough attention has been devoted to such large edifices beyond the basic identification of secondary structures. The objective of the present study is to show that the association of secondary structures that share some of their backbone residues is a commonplace in globular proteins, and that such deeper fusion of secondary structures, namely extended secondary structures (ESSs), helps stabilize the original secondary structures and the resulting tertiary structures. For statistical purposes, a set of 163 proteins from the protein databank was randomly selected and a few specific cases are structurally analyzed and characterized in more detail. The results point that about 30%of the residues from each protein, on average, participate in ESS. Alternatively, for the specific cases considered,our results were based on the secondary structures produced after extensive Molecular Dynamics simulation of a protein–aqueous solvent system. Based on the very small width of the time distribution of the root mean squared deviations, between the ESS taken along the simulation and the ESS from the mean structure of the protein, for each ESS, we conclude that the ESSs significantly increase the conformational stability by forming very stable aggregates.The ubiquity and specificity of the ESS suggest that the role they play in the structure of proteins, including the domains formation, deserves to be thoroughly investigated.

Singlet oxygen production in intact cells of the cynobacterium Synechocystis 6803 was studied using chemical trapping by histidine, which leads to O2 uptake during illumination. The rate of O2 uptake, measured by a standard Clark-type electrode, is enhanced in the presence of D2O, which increases the lifetime of (1)O2, and suppressed by the (1)O2 quencher NaN3. Due to the limited mobility of (1)O2 these data demonstrate that exogenous histidine reaches close vicinity of (1)O2 production sites inside the cells. Flash induced chlorophyll fluorescence measurements showed that histidine does not inhibit Photosystem II activity up to 5mM concentration. By applying the histidine-mediated O2 uptake method we showed that (1)O2 production linearly increases with light intensity even above the saturation of photosynthesis. We also studied (1)O2 production in site directed mutants in which the Gln residue at the 130th position of the D1 reaction center subunit was changed to either Glu or Leu, which affect the efficiency of nonradiative charge recombination from the primary radical pair (Rappaport et al. 2002, Biochemistry 41: 8518-8527; Cser and Vass 2007, BBA 1767:233-243). We found that the D1-Gln130Glu mutant showed decreased (1)O2 production concomitant with decreased rate of photodamage relative to the WT, whereas both (1)O2 production and photodamage were enhanced in the D1-Gln130Leu mutant. The data are discussed in the framework of the model of photoinhibition in which (3)P680 mediated (1)O2 production plays a key role in PSII photodamage, and nonradiative charge recombination of the primary charge separated state provides a photoprotective pathway., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The objective of this experiment was to evaluate the effect of supplementing a metabolizable protein (MP)-deficient diet with rumen-protected (RP) Lys, Met, and specifically His on dairy cow performance. The experiment was conducted for 12 wk with 48 Holstein cows. Following a 2-wk covariate period, cows were blocked by DIM and milk yield and randomly assigned to 1 of 4 diets, based on corn silage and alfalfa haylage: control, MP-adequate diet (ADMP; MP balance: +9 g/d); MP-deficient diet (DMP; MP balance: -317 g/d); DMP supplemented with RPLys (AminoShure-L, Balchem Corp., New Hampton, NY) and RPMet (Mepron; Evonik Industries AG, Hanau, Germany; DMPLM); and DMPLM supplemented with an experimental RPHis preparation (DMPLMH). The analyzed crude protein content of the ADMP and DMP diets was 15.7 and 13.5 to 13.6%, respectively. The apparent total-tract digestibility of all measured nutrients, plasma urea-N, and urinary N excretion were decreased by the DMP diets compared with ADMP. Milk N secretion as a proportion of N intake was greater for the DMP diets compared with ADMP. Compared with ADMP, dry matter intake (DMI) tended to be lower for DMP, but was similar for DMPLM and DMPLMH (24.5, 23.0, 23.7, and 24.3 kg/d, respectively). Milk yield was decreased by DMP (35.2 kg/d), but was similar to ADMP (38.8 kg/d) for DMPLM and DMPLMH (36.9 and 38.5kg/d, respectively), paralleling the trend in DMI. The National Research Council 2001model underpredicted milk yield of the DMP cows by an average (±SE) of 10.3 ± 0.75 kg/d. Milk fat and true protein content did not differ among treatments, but milk protein yield was increased by DMPLM and DMPLMH compared with DMP and was not different from ADMP. Plasma essential amino acids (AA), Lys, and His were lower for DMP compared with ADMP. Supplementation of the DMP diets with RP AA increased plasma Lys, Met, and His. In conclusion, MP deficiency, approximately 15% below the National Research Council requirements from 2001, decreased DMI and milk yield in dairy cows. Supplementation of the MP-deficient diet with RPLys and RPMet diminished the difference in DMI and milk yield compared with ADMP and additional supplementation with RPHis eliminated it. As total-tract fiber digestibility was decreased with the DMP diets, but DMI tended to increase with RP AA supplementation, we propose that, similar to monogastric species, AA play a role in DMI regulation in dairy cows. Our data implicate His as a limiting AA in high-producing dairy cows fed corn silage- and alfalfa haylage-based diets, deficient in MP. The MP-deficient diets clearly increased milk N efficiency and decreased dramatically urinary N losses., (Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Study Objectives: Neuronal histamine shows diurnal rhythms in rodents and plays a major role in the maintenance of vigilance. No data are available on its diurnal fluctuation in humans, either in health or in neurodegenerative disorders such as Parkinson disease (PD), Alzheimer disease (AD), or Huntington disease (HD), all of which are characterized by sleep-wake disturbances., Design: Quantitative in situ hybridization was used to study the mRNA expression of histidine decarboxylase (HDC), the key enzyme of histamine production in the tuberomammillary nucleus (TMN) in postmortem human hypothalamic tissue, obtained from 33 controls and 31 patients with a neurodegenerative disease-PD (n = 15), AD (n = 9), and HD (n = 8)-and covering the full 24-h cycle with respect to clock time of death., Results: HDC-mRNA levels in controls were found to be significantly higher during the daytime than at night (e.g., 08:01-20:00 versus 20:01-08:00, P = 0.004). This day-night fluctuation was markedly different in patients with neurodegenerative diseases., Conclusion: The diurnal fluctuation of HDC-mRNA expression in human TMN supports a role for neuronal histamine in regulating day-night rhythms. Future studies should investigate histamine rhythm abnormalities in neurodegenerative disorders., Citation: Shan L; Hofman MA; van Wamelen DJ; Van Someren EJW; Bao AM; Swaab DF. Diurnal fluctuation in histidine decarboxylase expression, the rate limiting enzyme for histamine production, and its disorder in neurodegenerative diseases.

Chloride intracellular channel proteins exist in both a soluble cytosolic form and a membrane-bound form. The mechanism of conversion between the two forms is not properly understood, although one of the contributing factors is believed to be the variation in pH between the cytosol (~7.4) and the membrane (~5.5). We systematically mutated each of the three histidine residues in CLIC1 to an alanine at position 74 and a phenylalanine at positions 185 and 207. We examined the effect of the histidine-mediated pH dependence on the structure and global stability of CLIC1. None of the mutations were found to alter the global structure of the protein. However, the stability of H74A-CLIC1 and H185F-CLIC1, as calculated from the equilibrium unfolding data, is no longer dependent on pH because similar trends are observed at pH 7.0 and 5.5. The crystal structures show that the mutations result in changes in the local hydrogen bond coordination. Because the mutant total free energy change upon unfolding is not different from that of the wild type at pH 7.0, despite the presence of intermediates that are not seen in the wild type, we propose that it may be the stability of the intermediate state rather than the native state that is dependent on pH. On the basis of the lower stability of the intermediate in the H74A and H185F mutants compared to that of the wild type, we conclude that both His74 and His185 are involved in triggering the pH changes to the conformational stability of wild-type CLIC1 via their protonation, which stabilizes the intermediate state.

Pyridoxal 5'-phosphate (PLP), in the active site of ornithine 4,5-aminomutase (OAM), forms a Schiff base with N(δ) of the d-ornithine side chain and facilitates interconversion of the amino acid to (2R, 4S) 2,4-diaminopentanoic acid via a radical-based mechanism. The crystal structure of OAM reveals that His225 is within hydrogen bond distance to the PLP phenolic oxygen, and may influence the pK(a) of the Schiff base during radical rearrangement. To evaluate the role of His225 in radical stabilization and catalysis, the residue was substituted with a glutamine and alanine. The H225Q and H225A variants have a 3- and 10-fold reduction in catalytic turnover, respectively, and a decrease in catalytic efficiency (7-fold for both mutants). Diminished catalytic performance is not linked to an increase in radical-based side reactions leading to enzyme inactivation. pH-dependence studies show that k(cat) increases with the ionization of a functional group, but it is not attributed to His225. Binding of 2,4-diaminobutyric acid to native OAM leads to formation of an overstabilized 2,4-diaminobutyryl-PLP derived radical. In the H225A and the H225Q mutants, the radical forms and then decays, as evidenced by accumulation of cob(III)alamin. From these data, we propose that His225 enhances radical stability by acting as a hydrogen bond acceptor to the phenolic oxygen, which favors the deprotonated state of the imino nitrogen and leads to greater resonance stabilization of the 2,4-diaminobutyryl-PLP radical intermediate. The potential role of His225 in lowering the activation energy barrier to mediate PLP-dependent radical rearrangement is discussed., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The first component (E1o) of the Escherichia coli 2-oxoglutarate dehydrogenase complex (OGDHc) was engineered to accept substrates lacking the 5-carboxylate group by subjecting H260 and H298 to saturation mutagenesis. Apparently, H260 is required for substrate recognition, but H298 could be replaced with hydrophobic residues of similar molecular volume. To interrogate whether the second component would allow synthesis of acyl-coenzyme A derivatives, hybrid complexes consisting of recombinant components of OGDHc (o) and pyruvate dehydrogenase (p) enzymes were constructed, suggesting that a different component is the "gatekeeper" for specificity for these two multienzyme complexes in bacteria, E1p for pyruvate but E2o for 2-oxoglutarate.

The oligopeptide transporter PepT1 is a protein found in the membrane of the cells of the intestinal walls, and represents the main route through which proteic nutrients are absorbed by the organism. Along the polypeptidic chain of this protein, two oppositely charged amino acids, an arginine in position 282 and an aspartate in position 341 of the sequence, have been hypothesised to form a barrier in the absorption pathway. In this paper we show that appropriate mutations of these amino acids change the properties of PepT1 in a way that confirms that these parts of the protein indeed act as an electrostatic gate in the transport process. The identification of the structural basis of the functional mechanism of this transporter is important because, in addition to its role in nutrient uptake, PepT1 represents a major pathway for the absorption of several therapeutic drugs.

The M2 protein of influenza viruses forms an acid-activated tetrameric proton channel. We used solid-state nuclear magnetic resonance spectroscopy to determine the structure and functional dynamics of the pH-sensing and proton-selective histidine-37 in M2 bound to a cholesterol-containing virus-envelope-mimetic membrane so as to better understand the proton conduction mechanism. In the high-pH closed state, the four histidines form an edge-face π-stacked structure, preventing the formation of a hydrogen-bonded water chain to conduct protons. In the low-pH conducting state, the imidazoliums hydrogen-bond extensively with water and undergo microsecond ring reorientations with an energy barrier greater than 59 kilojoules per mole. This barrier is consistent with the temperature dependence of proton conductivity, suggesting that histidine-37 dynamically shuttles protons into the virion. We propose a proton conduction mechanism in which ring-flip-assisted imidazole deprotonation is the rate-limiting step.

Mechanosensitive K(+) channels TREK1 and TREK2 form a subclass of two P-domain K(+) channels. They are potently activated by polyunsaturated fatty acids and are involved in neuroprotection, anesthesia, and pain perception. Here, we show that acidification of the extracellular medium strongly inhibits TREK1 with an apparent pK near to 7.4 corresponding to the physiological pH. The all-or-none effect of pH variation is steep and is observed within one pH unit. TREK2 is not inhibited but activated by acidification within the same range of pH, despite its close homology with TREK1. A single conserved residue, H126 in TREK1 and H151 in TREK2, is involved in proton sensing. This histidine is located in the M1P1 extracellular loop preceding the first P domain. The differential effect of acidification, that is, activation for TREK2 and inhibition for TREK1, involves other residues located in the P2M4 loop, linking the second P domain and the fourth membrane-spanning segment. Structural modeling of TREK1 and TREK2 and site-directed mutagenesis strongly suggest that attraction or repulsion between the protonated side chain of histidine and closely located negatively or positively charged residues in P2M4 control outer gating of these channels. The differential sensitivity of TREK1 and TREK2 to external pH variations discriminates between these two K(+) channels that otherwise share the same regulations by physical and chemical stimuli, and by hormones and neurotransmitters.

A disagreement on the mode of histidine binding to copper and the structure of [Cu(2+)(His)(2)] in solution still exists. Spectroscopic data in solution support a six-coordinate species with N4O2 donor atoms, while X-ray crystallography reveals five-coordinate N(3)O(2) donor atoms. We modified [Cu(2+)(His)(2)] in solution using diethyl pyrocarbonate (DEPC) and monitored the products spectrophotometrically and by mass spectrometry. Our spectrophotometric study indicates the presence of a free imidazole in the [Cu(2+)(His)(2)] complex in solution. Mass spectral characterization of a DEPC-modified [Cu(2+)(His)(2)] complex yielded a peak at 587.8 amu corresponding to three DEPC adducts. Taken together, our data indicate that the [Cu(2+)(His)(2)] complex in solution exists as a neutral five-coordinate structure with N3O2 donor atoms.

Multidrug ABC transporters such as the human multidrug resistance P-glycoprotein (ABCB1) play an important role in the extrusion of drugs from the cell and their overexpression can be a cause of failure of anticancer and antimicrobial chemotherapy. These transport systems contain two nucleotide-binding domains (NBDs) where ATP is bound and hydrolyzed and two membrane domains (MDs) which mediate vectorial transport of substrates across the cell membrane. Recent crystal structures of the bacterial ABCB1 homologues Sav1866 from Staphylococcus aureus and MsbA from Salmonella typhimurium and other organisms shed light on the possible conformational states adopted by multidrug ABC transporters during transport. These structures help to interpret cellular and biochemical data gathered on these transport proteins over the past three decades. However, there are contradictory views on how the catalytic cycle of ATP binding and hydrolysis by the NBDs is linked to the change in drug binding affinity at the MDs, which underlies the capture (high affinity) of the transported drug on one side of the membrane and its release (low affinity) on the other. This review provides an overview of the current evidence for the different transport models and establishes the most recent structure-function relationships in multidrug ABC transporters.

GSTs (glutathione transferases) are an important class of enzymes involved in cellular detoxification. GSTs are found in all classes of organisms and are implicated in resistance towards drugs, pesticides, herbicides and antibiotics. The activity, structure and folding, particularly of eukaryotic GSTs, have therefore been widely studied. The crystal structure of EGST (GST from Escherichia coli) was reported around 10 years ago and it suggested Cys(10) and His(106) as potential catalytic residues. However, the role of these residues in catalysis has not been further investigated, nor have the folding properties of the protein been described. In the present study we investigated the contributions of residues Cys(10) and His(106) to the activity and stability of EGST. We found that EGST shows a complex equilibrium unfolding profile, involving a population of at least two partially folded intermediates, one of which is dimeric. Mutation of residues Cys(10) and His(106) leads to stabilization of the protein and affects the apparent steady-state kinetic parameters for enzyme catalysis. The results suggest that the imidazole ring of His(106) plays an important role in the catalytic mechanism of the enzyme, whereas Cys(10) is involved in binding of the substrate, glutathione. Engineering of the Cys(10) site can be used to increase both the stability and GST activity of EGST. However, in addition to GST activity, we discovered that EGST also possesses thiol:disulfide oxidoreductase activity, for which the residue Cys(10) plays an essential role. Further, tryptophan quenching experiments indicate that a mixed disulfide is formed between the free thiol group of Cys(10) and the substrate, glutathione.

The sodium/iodide symporter (SLC5A5; also known as NIS), a transmembrane glycoprotein principally in the thyroid gland, is responsible for the accumulation of iodide necessary for thyroid hormones. Our previous study indicated that a novel exon 6 deletion (residues 233-280) in SLC5A5 loses the iodide uptake activity. Herein we characterized the role of His-226 in iodide transport of SLC5A5. His-226, a highly conserved extracellular residue among SLC5A5 homologs, was replaced with alanine, aspartic acid, glutamic acid, or lysine. All the SLC5A5 mutants were expressed normally in the cells and targeted correctly to the plasma membrane. However, all of the mutants displayed severe defects in iodide uptake, suggesting that His-226 was critical for iodide uptake. Kinetic analysis further showed that mutation at His-226 led to a dramatic decrease in V(max). These findings suggested that the decreased levels of iodide uptake activity of SLC5A5 mutants resulted from lower catalytic rates. In conclusion, our data first identified the involvement of extracellular charged amino acid residue in the iodide uptake ability of SLC5A5.

Protein phosphorylation represents one of the key regulatory events in physiological insulin secretion from the islet beta-cell. In this context, several classes of protein kinases (e.g. calcium-, cyclic nucleotide- and phospholipid-dependent protein kinases and tyrosine kinases) have been characterized in the beta-cell. The majority of phosphorylated amino acids identified include phosphoserine, phosphothreonine and phosphotyrosine. Protein histidine phosphorylation has been implicated in the prokaryotic and eukaryotic cellular signal transduction. Most notably, phoshohistidine accounts for 6% of total protein phosphorylation in eukaryotes, which makes it nearly 100-fold more abundant than phosphotyrosine, but less abundant than phosphoserine and phosphothreonine. However, very little is known about the number of proteins with phosphohistidines, since they are highly labile and are rapidly lost during phosphoamino acid identification under standard experimental conditions. The overall objectives of this review are to: (i) summarize the existing evidence indicating the subcellular distribution and characterization of various histidine kinases in the islet beta-cell, (ii) describe evidence for functional regulation of these kinases by agonists of insulin secretion, (iii) present a working model to implicate novel regulatory roles for histidine kinases in the receptor-independent activation, by glucose, of G-proteins endogenous to the beta-cell, (iv) summarize evidence supporting the localization of protein histidine phosphatases in the islet beta-cell and (v) highlight experimental evidence suggesting potential defects in the histidine kinase signalling cascade in islets derived from the Goto-Kakizaki (GK) rat, a model for type 2 diabetes. Potential avenues for future research to further decipher regulatory roles for protein histidine phosphorylation in physiological insulin secretion are also discussed.

Towards understanding the catalytic mechanism of M.EcoP15I [EcoP15I MTase (DNA methyltransferase); an adenine methyltransferase], we investigated the role of histidine residues in catalysis. M.EcoP15I, when incubated with DEPC (diethyl pyrocarbonate), a histidine-specific reagent, shows a time- and concentration-dependent inactivation of methylation of DNA containing its recognition sequence of 5'-CAGCAG-3'. The loss of enzyme activity was accompanied by an increase in absorbance at 240 nm. A difference spectrum of modified versus native enzyme shows the formation of N-carbethoxyhistidine that is diminished by hydroxylamine. This, along with other experiments, strongly suggests that the inactivation of the enzyme by DEPC was specific for histidine residues. Substrate protection experiments show that pre-incubating the methylase with DNA was able to protect the enzyme from DEPC inactivation. Site-directed mutagenesis experiments in which the 15 histidine residues in the enzyme were replaced individually with alanine corroborated the chemical modification studies and established the importance of His-335 in the methylase activity. No gross structural differences were detected between the native and H335A mutant MTases, as evident from CD spectra, native PAGE pattern or on gel filtration chromatography. Replacement of histidine with alanine residue at position 335 results in a mutant enzyme that is catalytically inactive and binds to DNA more tightly than the wild-type enzyme. Thus we have shown in the present study, through a combination of chemical modification and site-directed mutagenesis experiments, that His-335 plays an essential role in DNA methylation catalysed by M.EcoP15I.

Combined quantum mechanical/molecular mechanical (QM/MM) simulations are carried out to analyze factors that dictate the proton transfer in carbonic anhydrase II (CAII), an enzyme that has been used as a prototypical example of long-range proton transfers in biomolecules. In contrast to the long-held conjecture in the experimental literature, the computed potentials of mean force (PMF) suggest that the proton transfer in CAII is not very sensitive to the orientation of the acceptor group (His 64) and, therefore, the number of water molecules that bridge the donor (zinc-water) and acceptor groups. Perturbative analysis indicates that a series of polar and charged residues close to the transfer pathways make the dominant contribution to the barrier and exothermicity of the proton transfer reaction, thus supporting the proposal from previous studies of Warshel and co-workers using a somewhat simpler QM/MM model that electrostatic interactions play a major role in the proton transfer in CAII. The PMF results are in striking contrast to previous analysis using the same QM/MM method but an ensemble of minimum energy path (MEP) calculations, which found a steep dependence of the barrier height on the number of bridging water molecules. Analysis of the configurations sampled in the PMF and MEP simulations suggests that this difference arises because the PMF simulations sample a largely stepwise mechanism while the local MEP calculations artificially favored concerted transfers due to the specific protocol used to generate the initial configurations. Therefore, this study presents a compelling argument for carrying out proper conformational sampling in the study of long-range proton transfers. Finally, we illustrate that Phi analysis, which has been widely used in protein folding studies, can potentially generate new mechanistic information for long-range proton transfers regarding the sequence of events. The results of the perturbation analysis and the Phi analysis provide opportunities for experimentally testing the mechanistic proposals from this study and our recent work in which a stepwise "proton hole" transfer pathway has been proposed.

Zolpidem is a positive allosteric modulator of GABA(A) receptors with sensitivity to subunit composition. While it acts with high affinity and efficacy at GABA(A) receptors containing the α(1) subunit, it has a lower affinity to GABA(A) receptors containing α(2) , α(3) , or α(5) subunits and has a very weak efficacy at receptors containing the α(5) subunit. Here, we show that replacing histidine in position 105 in the α(5) subunit by cysteine strongly stimulates the effect of zolpidem in receptors containing the α(5) subunit. The side chain volume of the amino acid residue in this position does not correlate with the modulation by zolpidem. Interestingly, serine is not able to promote the potentiation by zolpidem. The homologous residues to α(5) H105 in α(1) , α(2) , and α(3) are well-known determinants of the action of classical benzodiazepines. Other studies have shown that replacement of these histidines α(1) H101, α(2) H101, and α(3) H126 by arginine, as naturally present in α(4) and α(6) , leads to benzodiazepine insensitivity of these receptors. Thus, the nature of the amino acid residue in this position is not only crucial for the action of classical benzodiazepines but in α(5) containing receptors also for the action of zolpidem.

Staphylococcus aureus is the source of a large number of hospital-acquired infections, of which many are serious and can lead to death. Iron is critically important to the survival and growth of the bacterium, and complex, multistep mechanisms are present to fulfill the necessary iron requirement. Isd proteins located on the wall and membrane of S. aureus have been proposed to function in heme acquisition. We report characterization of the S. aureus heme-binding protein IsdE, the lipoprotein component of a membrane-localized ABC transporter that is believed key to receiving heme from cell wall-anchored Isd proteins. Magnetic circular dichroism (MCD) data, which greatly extend the results from our initial study of IsdE in bacterial cell lysates (Mack, J., Vermeiren, C., Heinrichs, D. E., and Stillman, M. J. (2004) Biochem. Biophys. Res. Commun. 320, 781-788), probe the ligand and redox properties of the bound heme. The MCD data show that IsdE, when overexpressed in E. coli, binds either ferric or ferrous heme but that the largest fraction is low spin ferrous heme. Studies of mutants allowed identification and characterization of the ligands in the fifth and sixth position on the heme iron as histidine, proximally, and methionine, distally. This histidine-methionine heme-iron ligation is unique to heme transport proteins. The smaller fraction of ferric heme in the protein is not bound by methionine, allowing for access by strong field ligands, such as cyanide. Electrospray ionization mass spectral data are reported for the first time and show that only one heme ligand binds per IsdE protein molecule. These data also show there is little change in the conformation of the protein between the heme-bound and heme-free species, indicating that the heme-free IsdE adopts a structure essentially independent of the heme. The mass spectral data clearly show that IsdE reversibly unwinds under denaturing conditions to form at least two distinct, heme-free conformations.

Zinc, iron and copper are concentrated in senile plaques of Alzheimer disease. Copper and iron catalyze the Fenton-Haber-Weiss reaction, which likely contributes to oxidative stress in neuronal cells. In this study, we found that ascorbate oxidase activity and the intensity of ascorbate radicals measured using ESR spectroscopy, generated by free Cu(II), was decreased in the presence of amyloid-beta (Abeta), the major component of senile plaques. Specifically, the ascorbate oxidase activity was strongly inhibited (85% decrease) in the presence of Abeta1-16 or Abeta1-42, whereas it was only slightly inhibited in the presence of Abeta1-12 or Abeta25-35 (<20% inhibition). Ascorbate-dependent hydroxyl radical generation by free Cu(II) decreased in the presence of Abeta in the identical order of Abeta1-42, Abeta1-16 > Abeta1-12 and was abolished in the presence of 2-fold molar excess glycylhystidyllysine (GHK). Ascorbate oxidase activity and ascorbate-dependent hydroxyl radical generation by free Fe(III) were inhibited by Abeta1-42, Abeta1-16, and Abeta1-12. Although Cu(II)-Abeta shows a significant SOD-like activity, the rate constant for the reaction of superoxide with Cu(II)-Abeta was much slower than that with SOD. Overall, our results suggest that His6, His13, and His14 residues of Abeta1-42 control the redox activity of transition metals present in senile plaques.

Sodium ascorbate and histidine were employed to protect fibrinogen against modifications followed by a gamma-irradiation process that could potentially inactivate the blood-borne viruses in plasma-derived products. Fibrinogen was irradiated (50 kGy total dose, on dry ice) using a 60Co source. Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot. Carbonyl groups were measured by the 2,4-dinitrophenylhydrazine-coupled method, and the fibrinogen clotting activity was assessed by different functional assays. In irradiated fibrinogen, the carbonyl group concentration was elevated three-fold versus control; and moderate fragmentation of largely Aalpha and Bbeta chains was revealed. The rate of thrombin-catalyzed fibrinogen polymerization was inhibited (average 50%) with normal fibrinopeptide release and with a minor decrease of total clottable fibrinogen and alpha-polymer formation. Ascorbate reduced the incorporation of carbonyls to the fibrinogen molecule (by > 50% at 50 mmol/l; P < 0.001). Contrary to ascorbate, which alone delayed the fibrinogen polymerization rate, histidine abolished irradiation-induced inhibition of fibrinogen polymerization (by 80% at 50 mmol/l; P < 0.001). In conclusion, even though ascorbate effectively protects fibrinogen from oxidation due to its adverse effects on fibrinogen function, it may not serve as a suitable radioprotective. On the contrary, the first definite evidence is provided that radiation-sterilized fibrinogen in the presence of histidine greatly retains its clotting capability.

This study examines the functional and structural effects of amino acid substitution at alpha(1)beta(2) interface of Hb Santa Clara (beta 97His-->Asn). We have characterized the variation by a combination of electrospray ionisation mass spectrometry and DNA sequence analysis followed by oxygen-binding experiments. Functional studies outlined an increased oxygen affinity, reduced effect of organic phosphates and a reduced Bohr effect with respect to HbA. In view of the primary role of this interface in the cooperative quaternary transition from the T to R conformational state, a theoretical three-dimensional model of Hb Santa Clara was generated. Structural investigations suggest that replacement of Asn for His beta 97 results in a significant stabilization of the high affinity R-state of the haemoglobin molecule with respect to the low affinity T-state. The role of beta FG4 position has been further examined by computational models of known beta FG4 variants, namely Hb Malmö (beta 97His-->Gln), Hb Wood (beta 97His-->Leu), Hb Nagoya (beta 97His-->Pro) and Hb Moriguchi (beta 97His-->Tyr). These findings demonstrate that, among the various residues at the alpha(1)beta(2) (and alpha(2)beta(1)) intersubunit interface, His beta FG4 contributes significantly to the quaternary constraints that are responsible for the low oxygen affinity of human deoxyhaemoglobin.

Scavenger receptor class B type I (SR-BI) facilitates the uptake of HDL cholesteryl esters (CEs) in a two-step process involving binding of HDL to its extracellular domain and transfer of HDL core CEs to a metabolically active membrane pool, where they are subsequently hydrolyzed by a neutral CE hydrolase. Recently, we characterized a mutant, G420H, which replaced glycine 420 in the extracellular domain of SR-BI with a histidine residue and had a profound effect on SR-BI function. The G420H mutant receptor exhibited a reduced ability to mediate selective HDL CE uptake and was unable to deliver HDL CE for hydrolysis, despite the fact that it retained the ability to bind HDL. This did not hold true if glycine 420 was replaced with an alanine residue; G420A maintained wild-type HDL binding and cholesterol transport activity. To further understand the role that glycine 420 plays in SR-BI function and why there was a disparity between replacing glycine 420 with a histidine versus an alanine, we generated a battery of point mutants by substituting glycine 420 with amino acids possessing side chains that were charged, hydrophobic, polar, or bulky and tested the resulting mutants for their ability to support HDL binding, HDL cholesterol transport, and delivery for hydrolysis. The results indicated that substitution with a negatively charged residue or a proline impaired cell surface expression of SR-BI or its interaction with HDL, respectively. Furthermore, substitution of glycine 420 with a positively charged residue reduced HDL CE uptake as well as its subsequent hydrolysis.

Murine leukemia virus (MLV) fusion is controlled by isomerization of the disulphide bond between the receptor-binding surface (SU) and fusion-active transmembrane subunits of the Env-complex. The bond is in SU linked to a CXXC motif. This carries a free thiol that upon receptor binding can be activated (ionized) to attack the disulphide and rearrange it into a disulphide isomer within the motif. To find out whether His8 in the conserved SPHQ sequence of Env directs thiol activation, we analyzed its ionization in MLV vectors with wtEnv and Env with His8 deleted or substituted for Tyr or Arg, which partially or completely arrests fusion. The ionization was monitored by following the pH effect on isomerization in vitro by Ca2+ depletion or in vivo by receptor binding. We found that wtEnv isomerized optimally at slightly basic pH whereas the partially active mutant required higher and the inactive mutants still higher pH. This suggests that His8 directs the ionization of the CXXC thiol.

The catalytic pentad of tyrosine recombinases, that assists the tyrosine nucleophile, includes a conserved histidine/tryptophan (His/Trp-III). Flp and Cre harbor tryptophan at this position; most of their kin recombinases display histidine. Contrary to the conservation rule, Flp(W330F) is a much stronger recombinase than Flp(W330H). The hydrophobicity of Trp330 or Phe330 is utilized in correctly positioning Tyr343 during the strand cleavage step of recombination. Why then is phenylalanine almost never encountered in the recombinase family at this conserved position? Using exogenous nucleophiles and synthetic methylphosphonate or 5'-thiolate substrates, we decipher that Trp330 also assists in the activation of the scissile phosphate and the departure of the 5'-hydroxyl leaving group. These two functions are consistent with the hydrogen bonding property of Trp330 as well as its location in structures of the Flp recombination complexes. However, van der Waals contact between Trp330 and Arg308 may also be important for the phosphate activation step. A structure based suppression strategy permits the inactive variant Flp(W330A) to be rescued by a second site mutation A339M. Modeling alanine and methionine at positions 330 and 339, respectively, in the Flp crystal structure suggests a plausible mechanism for active site restoration. Successful suppression suggests the possibility of evolving, by design, new active site configurations for tyrosine recombination.

Histidine-to-Aspartate (His-Asp) phosphorelay (or two-component) systems are common signal transduction mechanisms implicated in a wide variety of cellular responses to environmental stimuli in both prokaryotes and eukaryotes. For a model filamentous fungi, Aspergillus nidulans, in this study we first compiled a complete list of His-Asp phosphorelay components, including 15 genes for His-kinase (HK), four genes for response regulator (RR), and only one for histidine-containing phosphotransfer intermediate (HPt). For these RR genes, a set of deletion mutants was constructed so as to create a null allele for each. When examined these mutant strains under various conditions stressful for hyphal growth and asexual spore development, two of them (designated DeltasskA and DeltasrrA) showed a marked phenotype of hypersensitivity to oxidative stresses (particularly, to hydrogen peroxide). In this respect, expression of the vegetative-stage specific catB catalase gene was severely impaired in both mutants. Furthermore, conidia from DeltasskA were hypersensitive not only to treatment with H(2)O(2), but also to treatment at aberrantly low (4 degrees C) and high (50 degrees C) temperatures, resulting in reduced germination efficiency. In this respect, not only the catA catalase gene specific for asexual development, but also a set of genes encoding the enzymes for synthesis of certain stress tolerant compatible solutes, such as trehalose and glycerol, were markedly downregulated in conidia from DeltasskA. These results together are indicative of the physiological importance of the His-Asp phosphorelay signaling network involving the SskA and SrrA response regulators.

The imidazole (15)N signals of histidine 64 (His(64)), involved in the catalytic function of human carbonic anhydrase II (hCAII), were assigned unambiguously. This was accomplished by incorporating the labeled histidine as probes for solution NMR analysis, with (15)N at ring-N(delta1) and N(epsilon2), (13)Cat ring-Cepsilon1, (13)C and (15)N at all carbon and nitrogen, or (15)N at the amide nitrogen and the labeled glycine with (13)C at the carbonyl carbon. Using the pH dependence of ring-(15)N signals and a comparison between experimental and simulated curves, we determined that the tautomeric equilibrium constant (K(T)) of His(64) is 1.0, which differs from that of other histidine residues. This unique value characterizes the imidazole nitrogen atoms of His(64) as both a general acid (a) and base (b): its epsilon2-nitrogen as (a) releases one proton into the bulk, whereas its delta1-nitrogen as (b) extracts another proton from a water molecule within the water bridge coupling to the zinc-bound water inside the cave. This accelerates the generation of zinc-bound hydroxide to react with the carbon dioxide. Releasing the productive bicarbonate ion from the inside separates the water bridge pathway, in which the next water molecules move into beside zinc ion. A new water molecule is supplied from the bulk to near the delta1-nitrogen of His(64). These reconstitute the water bridge. Based on these features, we suggest here a catalytic mechanism for hCAII: the tautomerization of His(64) can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time- or energy-consuming processes.

Neurospora crassa has a putative histidine phosphotransfer protein (HPT-1) that transfers signals from 11 histidine kinases to two putative response regulators (RRG-1 and RRG-2) in its histidine-to-aspartate phosphorelay system. The hpt-1 gene was successfully disrupted in the os-2 (MAP kinase gene) mutant, but not in the wild-type strain in this study. Crossing the resultant hpt-1; os-2 mutants with the wild-type or os-1 (histidine kinase gene) mutant strains produced no progeny with hpt-1 or os-1; hpt-1 mutation, strongly suggesting that hpt-1 is essential for growth unless downstream OS-2 is inactivated. hpt-1 mutation partially recovered the osmotic sensitivity of os-2 mutants, implying the involvement of yeast Skn7-like RRG-2 in osmoregulation. However, the rrg-2 disruption did not change the osmotic sensitivity of the wild-type strain and the os-2 mutant, suggesting that rrg-2 did not participate in the osmoregulation. Both rrg-2 and os-2 single mutation slightly increased sensitivity to t-butyl hydroperoxide, and rrg-2 and hpt-1 mutations increased the os-2 mutant's sensitivity. Although OS-1 is considered as a positive regulator of OS-2 MAP kinase, our results suggested that HPT-1 negatively regulated downstream MAP kinase cascade, and that OS-2 and RRG-2 probably participate independently in the oxidative stress response in N. crassa.

Mutation of a conserved His-157 in the second pore loop of KtrB drastically reduced the activity of the K+ transporter from Synechocystis sp. strain PCC 6803. This result suggests that His-157 plays an essential role in the K+ transport activity of the transporter system.

The anaerobic metabolism of phenol proceeds via carboxylation to 4-hydroxybenzoate by a two-step process involving seven proteins and two enzymes ("biological Kolbe-Schmitt carboxylation"). MgATP-dependent phosphorylation of phenol catalyzed by phenylphosphate synthase is followed by phenylphosphate carboxylation. Phenylphosphate synthase shows similarities to phosphoenolpyruvate (PEP) synthase and was studied for the bacterium Thauera aromatica. It consists of three proteins and transfers the beta-phosphoryl from ATP to phenol; the products are phenylphosphate, AMP, and phosphate. We showed that protein 1 becomes phosphorylated in the course of the reaction cycle by [beta-(32)P]ATP. This reaction requires protein 2 and is severalfold stimulated by protein 3. Stimulation of the reaction by 1 M sucrose is probably due to stabilization of the protein(s). Phosphorylated protein 1 transfers the phosphoryl group to phenolic substrates. The primary structure of protein 1 was analyzed by nanoelectrospray mass spectrometry after CNBr cleavage, trypsin digestion, and online high-pressure liquid chromatography at alkaline pH. His-569 was identified as the phosphorylated amino acid. We propose a catalytic ping-pong mechanism similar to that of PEP synthase. First, a diphosphoryl group is transferred to His-569 in protein 1, from which phosphate is cleaved to render the reaction unidirectional. Histidine phosphate subsequently serves as the actual phosphorylation agent.

Diethylpyrocarbonate modification and site-directed mutagenesis studies of histidine-35 in HM-1 killer toxin (HM-1) have shown that a specific feature, the imidazole side chain of histidine-35, is essential for the expression of the killing activity. In subcellular localization experiments, wild-type HM-1 was in the membrane fraction of Saccharomyces cerevisiae BJ1824, but not the HM-1 analogue in which histidine-35 was replaced by alanine (H35A HM-1). Neither wild-type nor H35A HM-1 was detected in cellular fractions of HM-1-resistant yeast S. cerevisiae BJ1824 rhk1Delta : : URA3 and HM-1-insensitive yeast Candida albicans even after 1 h incubation. H35A HM-1 inhibited the activity of partially purified 1,3-beta-glucan synthase from S. cerevisiae A451, and its extent was almost the same as wild-type HM-1. Co-immunoprecipitation experiments showed that wild-type and H35A HM-1 directly interact with the 1,3-beta-glucan synthase complex. These results strongly suggest that histidine-35 has an important role in the cytocidal action of HM-1 that participates in the binding process to the HM-1 receptor protein on the cell membrane, but it is not essential for the interaction with, and inhibition of, 1,3-beta-glucan synthase.

Histidine (His) is an essential ingredient for protein synthesis and is required by all living organisms. In higher plants, although there is considerable evidence that His is essential for plant growth and survival, there is very little information as to whether it plays any specific role in plant development. Here, we present evidence for such a role of this amino acid in root development in Arabidopsis (Arabidopsis thaliana) from the characterization of a novel Arabidopsis mutant, hpa1, which has a very short root system and carries a mutation in one of the two Arabidopsis histidinol-phosphate aminotransferase (HPA) genes, AtHPA1. We have established that AtHPA1 encodes a functional HPA and that its complete knockout is embryo lethal. Biochemical analysis shows that the mutation in hpa1 only resulted in a 30% reduction in free His content and had no significant impact on the total His content. It did not cause any known symptoms of His starvation. However, the mutant displayed a specific developmental defect in root meristem maintenance and was unable to sustain primary root growth 2 d after germination. We have demonstrated that the root meristem failure in the mutant is tightly linked to the reduction in free His content and could be rescued by either exogenous His supplementation or AtHPA1 overexpression. Our results therefore reveal an important role of His homeostasis in plant development.

Phytochelatin (PC) synthases are gamma-glutamylcysteine (gamma-Glu-Cys) dipeptidyl transpeptidases that catalyze the synthesis of heavy metal-binding PCs, (gamma-Glu-Cys)nGly polymers, from glutathione (GSH) and/or shorter chain PCs. Here it is shown through investigations of the enzyme from Arabidopsis (Arabidopsis thaliana; AtPCS1) that, although the N-terminal half of the protein, alone, is sufficient for core catalysis through the formation of a single-site enzyme acyl intermediate, it is not sufficient for acylation at a second site and augmentative stimulation by free Cd2+. A purified N-terminally hexahistidinyl-tagged AtPCS1 truncate containing only the first 221 N-terminal amino acid residues of the enzyme (HIS-AtPCS1&#95;221tr) is competent in the synthesis of PCs from GSH in media containing Cd2+ or the synthesis of S-methyl-PCs from S-methylglutathione in media devoid of heavy metal ions. However, whereas its full-length hexahistidinyl-tagged equivalent, HIS-AtPCS1, undergoes gamma-Glu-Cys acylation at two sites during the Cd2+-dependent synthesis of PCs from GSH and is stimulated by free Cd2+ when synthesizing S-methyl-PCs from S-methylglutathione, HIS-AtPCS1&#95;221tr undergoes gamma-Glu-Cys acylation at only one site when GSH is the substrate and is not directly stimulated, but instead inhibited, by free Cd2+ when S-methylglutathione is the substrate. Through the application of sequence search algorithms capable of detecting distant homologies, work we reported briefly before but not in its entirety, it has been determined that the N-terminal half of AtPCS1 and its equivalents from other sources have the hallmarks of a papain-like, Clan CA Cys protease. Whereas the fold assignment deduced from these analyses, which substantiates and is substantiated by the recent determination of the crystal structure of a distant prokaryotic PC synthase homolog from the cyanobacterium Nostoc, is capable of explaining the strict requirement for a conserved Cys residue, Cys-56 in the case of AtPCS1, for formation of the biosynthetically competent gamma-Glu-Cys enzyme acyl intermediate, the primary data from experiments directed at determining whether the other two residues, His-162 and Asp-180 of the putative papain-like catalytic triad of AtPCS1, are essential for catalysis have yet to be presented. This shortfall in our basic understanding of AtPCS1 is addressed here by the results of systematic site-directed mutagenesis studies that demonstrate that not only Cys-56 but also His-162 and Asp-180 are indeed required for net PC synthesis. It is therefore established experimentally that AtPCS1 and, by implication, other eukaryotic PC synthases are papain Cys protease superfamily members but ones, unlike their prokaryotic counterparts, which, in addition to having a papain-like N-terminal catalytic domain that undergoes primary gamma-Glu-Cys acylation, contain an auxiliary metal-sensing C-terminal domain that undergoes secondary gamma-Glu-Cys acylation.

Viral movement proteins (MPs) are central to the establishment of viral pathogenesis, and yet relatively little is understood about the structural and functional aspects of MPs or about the host factors on which they depend. Through chemical mutagenesis of transgenic Arabidopsis expressing Cucumber mosaic virus (CMV) MP fused with the green fluorescent protein, we have studied the function of a central region of the MP, defined by a number of conserved cysteine and histidine residues (Cys-His-rich region), which potentially functions as a zinc-binding domain. Transient expression of mutant MPs identified through an in planta screen for altered MP function or constructed with altered putative zinc ligands through site-directed mutagenesis showed that mutations in the Cys-His-rich region affected localization to and trafficking through plasmodesmata. In vitro zinc-binding analysis revealed that wild type (wt) CMV MP had the ability to bind zinc and that movement-defective mutants bound zinc with less affinity than wt MP. Furthermore, a correlation between the association of the MP with plasmodesmata and virus pathogenesis was shown. We discuss roles of the Cys-His region in biochemical and biological functions of the MP during virus movement.

Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded antibiotic peptide consisting of 21 L-amino acid residues (G1-G-A-G-H5-V-P-E-Y-F10-V-G-I-G-T15-P-I-S-F-Y20-G). E. coli RNA polymerase (RNAP) is the intracellular target of MccJ25. MccJ25 enters cells after binding to specific membrane transporters: FhuA in the outer membrane and SbmA in the inner membrane. Here, we studied MccJ25 mutants carrying a substitution of His5 by Lys, Arg, or Ala. The inhibitory effects on cellular growth and in vitro RNAP activity were determined for each mutant microcin. The results show that all mutants inhibited RNAP in vitro. However, the mutants were defective in their ability to inhibit cellular growth. Experiments in which the FhuA protein was bypassed showed that substitutions of MccJ25 His5 affected the SbmA-dependent transport. Our results thus suggest that MccJ25 His5 located in the lariat ring is involved, directly or indirectly, in specific interaction with SbmA and is not required for MccJ25 inhibition of RNAP.

The shaking pup (shp) is a canine mutation that affects the myelin protein proteolipid protein (PLP) and its smaller and less abundant isoform, DM20, with proline replacing histidine(36), resulting in a severe myelin deficiency in the central nervous system. We present evidence that the mutation leads to disrupted trafficking of the shp PLP/DM20 within oligodendrocytes. Immunohistochemical studies revealed significantly reduced levels of PLP/DM20 and other major myelin components such as myelin basic protein (MBP), myelin associated glycoprotein (MAG), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in shp myelin. The distribution of shp PLP/DM20 proteins were altered and mostly retained in perinuclear cytoplasm and proximal processes, which co-localized with distended rough endoplasmic reticulum (RER) within oligodendrocytes. No abnormal accumulation of MAG, MBP, or CNP in the cell body was found. These results suggest that mutated PLP/DM20 in the shp could be selectively retained in RER, causing disruption of their translocation to the periphery to myelinate axons., ((c) 2005 Wiley-Liss, Inc.)

Bacteria often use anti-terminator proteins to sense a specific metabolite signal and direct RNA polymerase to either terminate transcription or transcribe the downstream genes of an operon. Although many proteins that regulate various operons using this mechanism have been identified, insights into their activation processes before cognate mRNA binding have remained obscure. HutP from Bacillus subtilis regulates the hut operon by an anti-termination mechanism. Recently, several crystal structures of HutP [apo-HutP, HutP-L-histidine (and histidine analog), HutP-L-histidine-Mg(2+) and HutP-L-histidine-Mg(2+)-RNA] have been reported. These structural and functional studies of HutP have revealed how the protein undergoes conformational changes in response to two key components: L-histidine and Mg(2+) ions.

As an effort to elucidate the quaternary structure of cyclomaltodextrinase I-5 (CDase I-5) as a function of pH and salt concentration, the dissociation/association processes of the enzyme were investigated under various pH and salt conditions. Previous crystallographic analysis of CDase I-5 indicated that it existed exclusively as a dodecamer at pH 7.0, forming an assembly of six 3D domain-swapped dimeric subunits. In the present study, analytical ultracentrifugation analysis suggested that CDase I-5 was present as a dimer in the pH range of 5.0-6.0, while the dodecameric form was predominant at pH values above 6.5. No dissociation of the dodecamer was observed at pH 7.0 and the above. Gel filtration chromatography showed that CDase I-5 dissociated into dimers at a rate of 8.58 x 10(-2) h(-1) at pH 6.0. A mutant enzyme with three histidine residues (H49, H89, and H539) substituted with valines dissociated into dimers faster than the wild-type enzyme at both pH 6.0 and 7.0. The tertiary structure indicated that the effect of pH on dissociation of the oligomer was mainly due to the protonation of H539. Unlike the pH-dependent process, the dissociation of wild-type CDase I-5 proceeded very fast at pH 7.0 in the presence of 0.2-1.0 M of KCl. Stopped-flow spectrophotometric analysis at various concentrations of KCl showed that the rate constants of dissociation (kd) from dodecamers into dimers were 5.96 s(-1) and 7.99 s(-1) in the presence of 0.2 M and 1.0 M of KCl, respectively.

Plants exposed to heavy metals accumulate an array of metabolites, some to high millimolar concentrations. This review deals with N-containing metabolites frequently preferentially synthesized under heavy metal stress such as Cd, Cu, Ni, and Zn. Special focus is given to proline, but certain other amino acids and oligopeptides, as well as betaine, polyamines, and nicotianamine are also addressed. Particularly for proline a large body of data suggests significant beneficial functions under metal stress. In general, the molecules have three major functions, namely metal binding, antioxidant defence, and signalling. Strong correlative and mechanistic experimental evidence, including work with transgenic plants and algae, has been provided that indicates the involvement of metal-induced proline in metal stress defence. Histidine, other amino acids and particularly phytochelatins and glutathione play a role in metal binding, while polyamines function as signalling molecules and antioxidants. Their accumulation needs to be considered as active response and not as consequence of metabolic dys-regulation.

One of the most fundamental and widespread mechanisms of signal perception/transduction in prokaryotes is generally referred to as the "two-component regulatory system (TCS)." The concept of TCS has already been introduced a decade ago from extensive studies on the model prokaryotic bacterium Escherichia coli. Results of recent studies on the model higher plant Arabidopsis thaliana have led us to learn a new scenario as to the versatility of TCS in eukaryotic species. In the plant, on the one hand, TCS are crucially involved in the signal transduction mechanism underlying the regulation of sophisticated plant development in response to hormones (e.g., cytokinin and ethylene). On the other hand, a unique TCS variant is essentially integrated into the plant clock function that generates circadian rhythms, and also tells us the time and season on this regularly spinning and revolving world. In this review, recent progress with regard to studies on TCS in higher plants will be discussed, focusing particularly on the model higher plant Arabidopsis thaliana.

Heme A, as a prosthetic group, is found exclusively in respiratory oxidases of mitochondria and aerobic bacteria. Bacillus subtilis CtaA and other heme A synthases catalyze the conversion of a methyl side group on heme O into a formyl group. The catalytic mechanism of heme A synthase is not understood, and little is known about the composition and structure of the enzyme. In this work, we have: (i) constructed a ctaA deletion mutant and a system for overproduction of mutant variants of the CtaA protein in B. subtilis, (ii) developed anaffinity purification procedure for isolation of preparative amounts of CtaA, and (iii) investigated the functional roles of four invariant histidine residues in heme A synthase by in vivo and in vitro analyses of the properties of mutant variants of CtaA. Our results show an important function of three histidine residues for heme A synthase activity. Several of the purified mutant enzyme proteins contained tightly bound heme O. One variant also contained trapped hydroxylated heme O, which is a postulated enzyme reaction intermediate. The findings indicate functional roles for the invariant histidine residues and provide strong evidence that the heme A synthase enzyme reaction includes two consecutive monooxygenations.

L-Histidine (histidine), a precursor of neuronal histamine, has recently been hypothesized to suppress food intake. The association between dietary histidine and energy intake was examined among 1,689 Japanese female students of dietetic courses aged 18 y. Nutrient intakes were assessed over a 1-mo period with a validated, self-administered, diet history questionnaire. Both intake of histidine and the ratio of histidine to protein (histidine/ protein) statistically and positively correlated with energy intake. After adjustment for potential non-dietary confounding factors, including body height, body weight, physical activity level, and rate of eating, both the histidine intake and histidine/protein ratio statistically and positively correlated with energy intake (Pearson's correlation coefficient, r=0.62 and 0.12, respectively, p<0.001). Moreover, when protein or protein excluding histidine was additionally included into the covariates in order to minimize the effect of dietary factors and other amino acids, both histidine intake and histidine/protein ratio turned out to show a statistically negative correlation with energy intake (r=-0.22 and -0.23, respectively, p<0.001). Considering the influence of unavoidable various covariates, we found an inverse association between histidine/protein ratio and energy intake among the young female Japanese students.