A highly conserved His-His motif present in alpha1-->3/4fucosyltransferases is required for optimal activity and functions in acceptor binding

Alpha1-->3/4fucosyltransferases (FucTs) from several species contain a highly conserved His-His motif adjacent to an enzyme region correlating with the ability to catalyze fucose transfer to type 1 chain acceptors. Site-directed mutagenesis has been employed to analyze structure-function relationships of this His-His motif in human FucT-IV. The results indicate that most changes of His(113) and His(114) and nearby residues of FucT-IV reduced the specific activity of the enzymes. Analysis of acceptor properties demonstrated close similarity of most mutants with wild-type FucT-IV, whereas an apparent preference for the H-type II acceptor was observed for the His(114) mutants. Kinetic studies demonstrated that mutants of His(114) had a substantially increased K(m) for acceptor compared to other enzymes tested. The dramatic increase in acceptor K(m) for the His(114) mutants, particularly for the nonfucosylated acceptor, suggests that this His-His motif is involved in acceptor binding and perhaps interacts with GlcNAc residues of type 2 acceptors. The presence of fucose in acceptor substrates may promote more efficient substrate binding and presumably partially overcomes the weaker interaction with GlcNAc caused by the mutation.     

Prebiotic synthesis of histidine

Summary The prebiotic formation of histidine (His) has been accomplished experimentally by the reacton of erythrose with formamidine followed by a Strecker synthesis. In the first step of this reaction sequence, the formation of imidazole-4-acetaldehyde took place by the condensation of erythrose and formamidine, two compounds that are known to be formed under prebiotic conditions. In a second step, the imidazole-4-acetaldehyde was converted to His, without isolation of the reaction products by adding HCN and ammonia to the reaction mixture. LC, HPLC, thermospray liquid chromatography-mass spectrometry, and tandem mass spectrometry were used to identify the product, which was obtained in a yield of 3.5% based on the ratio of His/erythrose. This is a new chemical synthesis of one of the basic amino acids which has not been synthesized prebiotically until now.     

Prebiotic ribose synthesis: A critical analysis

The discovery of catalytic ability in RNA has given fresh impetus to speculations that RNA played a critical role in the origin of life. This question must rest on the plausibility of prebiotic oligonucleotide synthesis, rather than on the properties of the final product. Many cliams have been published to support the idea that the components of RNA were readily available on the prebiotic earth. In this article, the literature cited in support of the prebiotic availability of one subunit, D-ribose, is reviewed to determine whether it justifies the claim.Polymerization of formaldehyde (the formose reaction) has been the single reaction cited for prebiotic ribose synthesis. It has been conducted with different catalysts: numerous basic substances, neutral clays and heat, and various types of radiation. Ribose has been identified (yields are uncertain, but unlikely to be greater than 1%) in reactions run with concentrated (0.15 M or greater) formaldehyde. It has been claimed in reactions run at lower concentration, but characterization has been inadequate, and experimental details have not been provided.The complex sugar mixture produced in the formose reaction is rapidly destroyed under the reaction conditions. Nitrogenous substances (needed for prebiotic base synthesis) would interfere with the formose reaction by reacting with formaldehyde, the intermediates, and sugar products in undesirable ways.The evidence that is currently available does not support the availability of ribose on the prebiotic earth, except perhaps for brief periods of time, in low concentration as part of a complex mixture, and under conditions unsuitable for nucleoside synthesis.     

Converting a c-type to a b-type cytochrome: Met61 to His61 mutant of Pseudomonas cytochrome c-551

The gene nirM, coding for cytochrome c-551 in Pseudomonas stutzeri substrain ZoBell, was engineered to mutate Met61, the sixth ligand to the heme c, into His61, thereby converting the typical Met-His coordination of a c-type cytochrome into His-His, typical of b-type cytochromes. The mutant protein was expressed heterologously in Escherichia coli at levels 3-fold higher than in Pseudomonas and purified to homogeneity. The mutant retained low-spin visible spectral characteristics, indicating that the strong field ligand His 61 was coordinated to the iron. The physiochemical properties of the mutant were measured and compared to the wild-type properties. These included visible spectra, ligand binding reactions, stability to temperature and chemical denaturant, oxidation-reduction potentials, and electron-transfer kinetics to the physiological nitrite reductase of Pseudomonas. Despite a change in potential from the normal 260 mV to 55 mV, the mutant retained many of the properties of the c-551 family.     

Prebiotic synthesis of imidazole-4-acetaldehyde and histidine

The prebiotic synthesis of imidazole-4-acetaldehyde and imidazole-4-glycol from erythrose and formamidine has been demonstrated as well as the prebiotic synthesis of imidazole-4-ethanol and imidazole-4-glycol from erythrose, formaldehyde and ammonia. The products were identified by TLC, HPLC, and LC-MS by comparison with authentic samples. The maximum yields of imidazole-4-acetaldehyde, imidazole-4-ethanol, and imidazole-4-glycol obtained in these reactions are 1.6, 5.4, 6.8% respectively, based on the erythrose. Imidazole-4-acetaldehyde would have been converted to histidine on the primitive earth by a Strecker synthesis, and several prebiotic reactions would convert imidazole-4-glycol and imidazole-4-ethanol to imidazole-4-acetaldehyde.     

The lumenal loop Met672-Pro707 of copper-transporting ATPase ATP7A binds metals and facilitates copper release from the intramembrane sites

The copper-transporting ATPase ATP7A has an essential role in human physiology. ATP7A transfers the copper cofactor to metalloenzymes within the secretory pathway; inactivation of ATP7A results in an untreatable neurodegenerative disorder, Menkes disease. Presently, the mechanism of ATP7A-mediated copper release into the secretory pathway is not understood. We demonstrate that the characteristic His/Met-rich segment Met(672)-Pro(707) (HM-loop) that connects the first two transmembrane segments of ATP7A is important for copper release. Mutations within this loop do not prevent the ability of ATP7A to form a phosphorylated intermediate during ATP hydrolysis but inhibit subsequent dephosphorylation, a step associated with copper release. The HM-loop inserted into a scaffold protein forms two structurally distinct binding sites and coordinates copper in a mixed His-Met environment with an ～2:1 stoichiometry. Binding of either copper or silver, a Cu(I) analog, induces structural changes in the loop. Mutations of 4 Met residues to Ile or two His-His pairs to Ala-Gly decrease affinity for copper. Altogether, the data suggest a two-step process, where copper released from the transport sites binds to the first His(Met)(2) site, triggering a structural change and binding to a second 2-coordinate His-His or His-Met site. We also show that copper binding within the HM-loop stabilizes Cu(I) and protects it from oxidation, which may further aid the transfer of copper from ATP7A to acceptor proteins. The mechanism of copper entry into the secretory pathway is discussed.     

Selective Formation of Ser-His Dipeptide via Phosphorus Activation

The Ser-His dipeptide is the shortest active peptide. This dipeptide not only hydrolyzes proteins and DNA but also catalyzes the formation of peptides and phosphodiester bonds. As a potential candidate for the prototype of modern hydrolase, Ser-His has attracted increasing attention. To explore if Ser-His could be obtained efficiently in the prebiotic condition, we investigated the reactions of N-DIPP-Ser with His or other amino acids in an aqueous system. We observed that N-DIPP-Ser incubated with His can form Ser-His more efficiently than with other amino acids. A synergistic effect involving the two side chains of Ser and His is presumed to be the critical factor for the selectivity of this specific peptide formation.     

Geometry of interaction of the histidine ring with other planar and basic residues

Among the aromatic residues in protein structures, histidine (His) is unique, as it can exist in the neutral or positively charged form at the physiological pH. As such, it can interact with other aromatic residues as well as form hydrogen bonds with polar and charged (both negative and positive) residues. We have analyzed the geometry of interaction of His residues with nine other planar side chains containing aromatic (residues Phe, Tyr, Trp, and His), carboxylate (Asp and Glu), carboxamide (Asn and Gln) and guanidinium (Arg) groups in 432 polypeptide chains. With the exception of the aspartic (Asp) and glutamic (Glu) acid side-chains, all other residues prefer to interact in a face-to-face or offset-face-stacked orientation with the His ring. Such a geometry is different from the edge-to-face relative orientation normally associated with the aromatic-aromatic interaction. His-His pair prefers to interact in a face-to-face orientation; however, when both the residues bind the same metal ion, the interplanar angle is close to 90 degrees. The occurrence of different interactions (including the nonconventional N-H...pi and C-H...pi hydrogen bonds) have been correlated with the relative orientations between the interacting residues. Several structural motifs, mostly involved in binding metal ions, have been identified by considering the cases where His residues are in contact with four other planar moieties. About 10% of His residues used here are also found in sequence patterns in PROSITE database. There are examples of the amino end of the Lys side chain interacting with His residues in such a way that it is located on an arc around a ring nitrogen atom.     

Ab initio modelling of the structure and redox behaviour of copper(I) bound to a His–His model peptide: relevance to the β-amyloid peptide of Alzheimer’s disease

A contributing factor to the pathology of Alzheimer's disease is the generation of reactive oxygen species, most probably a consequence of the beta-amyloid (Abeta) peptide coordinating copper ions. Experimental and theoretical results indicate that His13 and His14 are the two most firmly established ligands in the coordination sphere of Cu(II) bound to Abeta. Abeta1-42 is known to reduce Cu(II) to Cu(I). The Abeta-Cu(II) complex has been shown to catalytically generate H(2)O(2) from reducing agents and O(2). Cu(II) in the presence of Abeta has been reported to have a formal reduction potential of +0.72-0.77 V (vs. the standard hydrogen electrode). Quantum chemical calculations using the B3LYP hybrid density functional method with the 6-31G(d) basis set were performed to model the reduction of previously studied Cu(II) complexes representing the His13-His14 portion of Abeta (Raffa et al. in J. Biol. Inorg. Chem. 10:887-902, 2005). The effects of solvation were accommodated using the CPCM method. The most stable complex between Cu(I) and the model compound, 3-(5-imidazolyl)propionylhistamine (1) involves tricoordinated Cu(I) in a distorted-T geometry, with the Npi of both imidazoles as well as the oxygen of the backbone carbonyl bound to copper. This model would be the most likely representation of a Cu(I) binding site for a His-His peptide in aqueous solution. A variety of possible redox processes are discussed.     

Evidence regarding the hypothesis that the histidine-histidine contact pairs may affect protein stability

It has been lately proposed that the interaction between like-charged residues stabilizes the native state of proteins. To explore this, we created a histidine-histidine pair in the Ca-III binding site of the Bacillus amyloliquefaciens α-amylase (BAA) and then examined the impact of this pairing on the BAA. For this purpose, we used site-directed mutagenesis (SDM) to substitute Pro407 with His, Ala, Gln, Arg, and Glu in the BAA. Subsequently, thermostability, kinetic parameters and structural properties of these variants were measured. Moreover, His-His pairing effect on the BAA thermostability was examined by simultaneous mutation of two residues (P407H/H406A and P407H/H406N). The data exhibited a significant improve in thermostability and structural features of enzyme by His replacement instead of Pro407. Other substitutions in this site did not have a significant effect on the enzyme properties, except for P407R, which yielded a partial improvement. The results also showed that the thermostabilities of double mutants significantly decreased compared with that of the P407H mutant. Moreover, the thermostability of P407H remarkably increased compared with that of other variants even in the absence of Ca(2+). Our data clearly demonstrated that His406-His407 pairing was the major cause for improved thermal stability.     

New evidence for a tubular structure ofβ-iron(III) oxide hydroxide — akaganéite

A biological method for the preparation of ultrathin transmission electron microscopy (TEM) sections has been used successfully to examine the fragile mineral akaganéite. TEM exposures reveal a tubular structure for akaganéite. The existence of such a structure has been debated for over a decade. The tubular structure may have been active in prebiotic polymerization reactions on the young Earth.     

Functional importance of tyrosine 294 and the catalytic selectivity for the bis-Fe(IV) state of MauG revealed by replacement of this axial heme ligand with histidine

The diheme enzyme MauG catalyzes the posttranslational modification of a precursor protein of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. It catalyzes three sequential two-electron oxidation reactions which proceed through a high-valent bis-Fe(IV) redox state. Tyr294, the unusual distal axial ligand of one c-type heme, was mutated to His, and the crystal structure of Y294H MauG in complex with preMADH reveals that this heme now has His-His axial ligation. Y294H MauG is able to interact with preMADH and participate in interprotein electron transfer, but it is unable to catalyze the TTQ biosynthesis reactions that require the bis-Fe(IV) state. This mutation affects not only the redox properties of the six-coordinate heme but also the redox and CO-binding properties of the five-coordinate heme, despite the 21 ? separation of the heme iron centers. This highlights the communication between the hemes which in wild-type MauG behave as a single diheme unit. Spectroscopic data suggest that Y294H MauG can stabilize a high-valent redox state equivalent to Fe(V), but it appears to be an Fe(IV)═O/π radical at the five-coordinate heme rather than the bis-Fe(IV) state. This compound I-like intermediate does not catalyze TTQ biosynthesis, demonstrating that the bis-Fe(IV) state, which is stabilized by Tyr294, is specifically required for this reaction. The TTQ biosynthetic reactions catalyzed by wild-type MauG do not occur via direct contact with the Fe(IV)═O heme but via long-range electron transfer through the six-coordinate heme. Thus, a critical feature of the bis-Fe(IV) species may be that it shortens the electron transfer distance from preMADH to a high-valent heme iron.     

Potentially prebiotic syntheses of condensed phosphates

In view of the importance of a prebiotic source of high energy phosphates, we have investigated a number of potentially prebiotic processes to produce condensed phosphates from orthophosphate and cyclic trimetaphosphate from tripolyphosphate. The reagents investigated include polymerizing nitriles, acid anhydrides, lactones, hexamethylene tetramine and carbon suboxide. A number of these processes give substantial yields of pyrophosphate from orthophosphate and trimetaphosphate from tripolyphosphate. Although these reactions may have been applicable in local areas, they are not sufficiently robust to have been of importance in the prebiotic open ocean.     

Interaction of Ribonucleotides with Metal Hexacyanocobaltate(III): A Possible Role in Chemical Evolution

It has been proposed that metal cyanide complexes would have acted as effective prebiotic catalysts. Insoluble metal cyanide complexes could have concentrated biomonomers from the dilute prebiotic soup, facilitating certain prebiotic reactions. In the light of the above hypothesis, interaction of four ribonucleotides, namely 5′-AMP, 5′-GMP, 5′-CMP, and 5′-UMP with copper(II)- and cadmium(II) hexacyanocobaltate(III) has been studied. The interaction was found to be maximum at neutral pH. 5′-GMP showed greater interaction with both the metal hexacyanocobaltate(III) while copper(II) hexacyanocobaltate(III) showed greater uptake than cadmium(II) hexacyanocobaltate(III) for all the four ribonucleotides studied. Infrared spectral studies of ribonucleotides, metal hexacyanocobaltate(III) and ribonucleotide – metal hexacyanocobaltate(III) adducts indicated that the nitrogen base and phosphate moiety of ribonucleotides interact with outer divalent metal ion present in the lattice of metal hexacyanocobaltate(III).     

Condensation reaction of hexanucleotides containing guanine and cytosine with water soluble carbodiimide.

The condensation reactions of hexanucleotides involving guanine and cytosine in the presence of water-soluble carbodiimide (WSC) have been investigated as a model reaction of the prebiotic formation of RNA under primitive earth. The reactions formed cyclic hexanucleotides and dimers in which the product yields were dependent on the sequence.     

Can an Imidazole Be Formed from an Alanyl-Seryl-Glycine Tripeptide under Possible Prebiotic Conditions?

The five-membered heterocyclic imidazole group, which is an essential component of purines, histidine and many cofactors, has been abiotically synthesized in different model experiments that attempt to simulate the prebiotic environment. The evolutionary significance of imidazoles is highlighted not only by its presence in nucleic acid components and in histidine, but also by experimental reports of its ability to restore the catalytic activity of ribozymes. However, as of today there are no reports of histidine in carbonaceous chondrites, and although the abiotic synthesis of His reported by Shen et al. (1987, 1990a) proceeds via an Amadori rearrangement, like in the biosynthesis of histidine, neither the reactants nor the conditions are truly prebiotic. Based on the autocatalytic biosynthesis of 4-methylidene-imidazole-one (MIO), a cofactor of some members of the amino acid aromatic ammonia-lyases and aminomutases, which occur via the self-condensation of a simple Ala-Ser-Gly motif within the sequence of the enzymes, we propose a possible prebiotic synthesis of an imidazolide.     

The Potential of Photochemical Transition Metal Reactions in Prebiotic Organic Synthesis. I. Observed Conversion of Methanol into Ethylene Glycol as Possible Prototype for Sugar Alcohol Formation

Photochemical processes involving redox reactions between metal ions and organic substrates possess the versatile potential for having harnessed solar energy for prebiotic organic synthesis. The present study in our Laboratory has shown that ultraviolet irradiation of transition metal ions such as of Ni, Co, Fe, Cu and Ti dissolved in primary or secondary alcohols causes photoreduction of the metal ions with the concomitant oxidation of the alcohol to aldehyde or ketone. An observed accompaniment of this novel 'light' reaction has been the known 'dark' pinacol reaction, whereby the carbonyl derivative underwent bimolecular coupling to the diol by the photogenerated reduced transition metal reagent. These tandem 'light-dark' processes possess the potential for the stepwise synthesis of dimeric 1,2-diols from simpler alcohols under conditions that might have prevailed on the prebiotic earth. Experiments reported here have demonstrated that such a tandem 'light-dark' conversion of methanol into ethylene glycol, via formaldehyde, does in fact occur, when nickel(II) acetylacetonate solutions in methanol undergo prolonged irradiation at 185-254 nm. Since ethylene glycol can be considered as the simplest sugar alcohol, these findings may provide novel insight into the prebiotic oligomerization of formaldehyde into higher sugar alcohols or even sugars.     

The distinct heme coordination environments and heme-binding stabilities of His39Ser and His39Cys mutants of cytochrome b5

A gene mutant library containing 16 designed mutated genes at His39 of cytochrome b(5) has been constructed by using gene random mutagenesis. Two variants of cytochrome b(5), His39Ser and His39Cys mutant proteins, have been obtained. Protein characterizations and reactions were performed showing that these two mutants have distinct heme coordination environments: ferric His39Ser mutant is a high-spin species whose heme is coordinated by proximal His63 and likely a water molecule in the distal pocket, while ferrous His39Ser mutant has a low-spin heme coordinated by His63 and Ser39; on the other hand, the ferric His39Cys mutant is a low-spin species with His63 and Cys39 acting as two axial ligands of the heme, the ferrous His39Cys mutant is at high-spin state with the only heme ligand of His63. These two mutants were also found to have quite lower heme-binding stabilities. The order of stabilities of ferric proteins is: wild-type cytochrome b(5) > His39Cys > His39Ser.     

Aminomalononitrile: Some new data of prebiotic interest

Malonic nitriles are of great interest in prebiotic chemistry, because of the various organic reactions they can give in aqueous solutions. Among those, aminomalononitrile (AMN) plays a key role, as the trimer of HCN. In order to determine some of the thermodynamical data (pKA),...) and kinetic data relating to AMN in water, kinetic studies of the behaviour of AMN in aqueous solutions has been carried out at various pH.The preliminary results are reported concerning the determination of:Acid-base dissociation constants and U.V. andabsorption coefficients     

Abiotic synthesis of purines and other heterocyclic compounds by the action of electrical discharges

Summary The synthesis of purines and pyrimidines using Oparin-Urey-type primitive Earth atmospheres has been demonstrated by reacting methane, ethane, and ammonia in electrical discharges. Adenine, guanine, 4-aminoimidazole-5-carboxamide (AICA), and isocytosine have been identified by UV spectrometry and paper chromatography as the products of the reaction. The total yields of the identified heterocyclic compounds are 0.0023%. It is concluded that adenine synthesis occurs at a much lower concentration of hydrogen cyanide than has been shown by earlier studies. Pathways for the synthesis of purines from hydrogen cyanide are discussed, and a comparison of the heterocyclic compounds that have been identified in meteorites and in prebiotic reactions is presented.