Schiff Base Intermediate Research Articles

Truncation of Amino-terminal Tail Stimulates Activity of Human Endonuclease III (hNTH1)

The human base excision repair enzyme hNTH1, a homologue of Escherichia coli endonuclease III (Nth), is a 36 kDa DNA glycosylase with associated abasic (AP) lyase activity. It has significant sequence homology with Nth in its DNA-binding motifs and catalytic residues but possesses a unique amino (N)-terminal tail (residues 1–95). We investigated the structure and function of this tail. Controlled proteolysis cleaved hNTH1 into discrete fragments to generate a 25 kDA core domain lacking the N-terminal 98 residues. Surprisingly, recombinant hNTH1 lacking 55, 72 or 80 residues from the N terminus had four- to fivefold higher activities than the full-length enzyme. Kinetic analysis at transition states revealed that release of the final product, an AP site with a 3′-nick, is the rate-limiting step in the multi-step reaction mediated by hNTH1. The N-terminal tail regulates its overall catalytic turnover by reducing this product release rate by five- to sevenfold without affecting either the glycosylase or AP lyase activities, or the steady-state equilibrium concentration of Schiff base intermediate, the covalent complex of hNTH1 and AP-site DNA formed after the base is excised. The inhibitory role of the N-terminal tail in catalytic turnover explains the low activity of hNTH1 compared to that of its E. coli homologue.

Proton transfers in the beta-reaction catalyzed by tryptophan synthase.

Reactions catalyzed by the beta-subunits of the tryptophan synthase alpha(2)beta(2) complex involve multiple covalent transformations facilitated by proton transfers between the coenzyme, the reacting substrates, and acid-base catalytic groups of the enzyme. However, the UV/Vis absorbance spectra of covalent intermediates formed between the pyridoxal 5'-phosphate coenzyme (PLP) and the reacting substrate are remarkably pH-independent. Furthermore, the alpha-aminoacrylate Schiff base intermediate, E(A-A), formed between L-Ser and enzyme-bound PLP has an unusual spectrum with lambda(max) = 350 nm and a shoulder extending to greater than 500 nm. Other PLP enzymes that form E(A-A) species exhibit intense bands with lambda(max) approximately 460-470 nm. To further investigate this unusual tryptophan synthase E(A-A) species, these studies examine the kinetics of H(+) release in the reaction of L-Ser with the enzyme using rapid kinetics and the H(+) indicator phenol red in solutions weakly buffered by substrate L-serine. This work establishes that the reaction of L-Ser with tryptophan synthase gives an H(+) release when the external aldimine of L-Ser, E(Aex(1)), is converted to E(A-A). This same H(+) release occurs in the reaction of L-Ser plus the indole analogue, aniline, in a step that is rate-determining for the appearance of E(Q)(Aniline). We propose that the kinetic and spectroscopic properties of the L-Ser reaction with tryptophan synthase reflect a mechanism wherein the kinetically detected proton release arises from conversion of an E(Aex(1)) species protonated at the Schiff base nitrogen to an E(A-A) species with a neutral Schiff base nitrogen. The mechanistic and conformational implications of this transformation are discussed.

Evidence for multiple imino intermediates and identification of reactive nucleophiles in peptide-catalyzed beta-elimination at abasic sites.

Prior investigations have demonstrated that peptides containing a single aromatic residue flanked by basic ones, such as Lys-Trp-Lys, can incise the phosphodiester backbone of duplex DNA at an AP site via beta-elimination. An amine serves as the reactive nucleophile to attack C1' on the ring-open deoxyribose sugar to form a transient peptide-DNA imino (Schiff base) intermediate, which may be isolated as a stable covalent species under reducing conditions. In the current study, we use this methodology to demonstrate that peptide-catalyzed beta-elimination proceeds via the formation of two Schiff base intermediates, one of which was covalently trapped prior to strand incision and the other following strand incision. N-Terminal acetylation of reactive peptides significantly inhibited formation of a trapped Schiff base complex; thus, we demonstrate for the first time that the preferred reactive nucleophile for peptides catalyzing strand incision is the N-terminal alpha-amino group, not an epsilon-amino group located on a lysine residue as previously postulated. Trapping reactions in which the central tryptophan residue was changed to alanine did not have a significant impact on the efficiency of Schiff base formation, indicating that the presence of an aromatic residue is dispensable for the step prior to peptide-catalyzed beta-elimination. Interestingly, the methodology presented here affords a convenient means for covalently attaching an array of peptides onto AP site-containing DNA in a site-specific fashion. We suggest that the generation of such DNA-peptide cross-links may provide utility in studying the repair of biologically significant DNA-protein cross-link damage as DNA-peptide complexes may mimic intermediate structures along a repair pathway for DNA-protein cross-links.

Synthesis of asymmetric derivatives of 1,4,7-triazacyclononane and trigonal prismatic Mn(ii) complexes

A new route to the asymmetric functionalisation of [9]aneN3 has been achieved via the use of macropolycyclic Schiff-base intermediates; novel trigonal prismatic Mn(II) complexes of these asymmetric ligands have been synthesised and structurally characterised.

Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate.

Endonuclease VIII (Nei) of Escherichia coli is a DNA repair enzyme that excises oxidized pyrimidines from DNA. Nei shares with formamidopyrimidine-DNA glycosylase (Fpg) sequence homology and a similar mechanism of action: the latter involves removal of the damaged base followed by two sequential beta-elimination steps. However, Nei differs significantly from Fpg in substrate specificity. We determined the structure of Nei covalently crosslinked to a 13mer oligodeoxynucleotide duplex at 1.25 A resolution. The crosslink is derived from a Schiff base intermediate that precedes beta-elimination and is stabilized by reduction with NaBH(4). Nei consists of two domains connected by a hinge region, creating a DNA binding cleft between domains. DNA in the complex is sharply kinked, the deoxyribitol moiety is bound covalently to Pro1 and everted from the duplex into the active site. Amino acids involved in substrate binding and catalysis are identified. Molecular modeling and analysis of amino acid conservation suggest a site for recognition of the damaged base. Based on structural features of the complex and site-directed mutagenesis studies, we propose a catalytic mechanism for Nei.

A Post-Amadori Inhibitor Pyridoxamine Also Inhibits Chemical Modification of Proteins by Scavenging Carbonyl Intermediates of Carbohydrate and Lipid Degradation

Reactive carbonyl compounds are formed during autoxidation of carbohydrates and peroxidation of lipids. These compounds are intermediates in the formation of advanced glycation end products (AGE) and advanced lipoxidation end products (ALE) in tissue proteins during aging and in chronic disease. We studied the reaction of carbonyl compounds glyoxal (GO) and glycolaldehyde (GLA) with pyridoxamine (PM), a potent post-Amadori inhibitor of AGE formation in vitro and of development of renal and retinal pathology in diabetic animals. PM reacted rapidly with GO and GLA in neutral, aqueous buffer, forming a Schiff base intermediate that cyclized to a hemiaminal adduct by intramolecular reaction with the phenolic hydroxyl group of PM. This bicyclic intermediate dimerized to form a five-ring compound with a central piperazine ring, which was characterized by electrospray ionization-liquid chromatography/mass spectrometry, NMR, and x-ray crystallography. PM also inhibited the modification of lysine residues and loss of enzymatic activity of RNase in the presence of GO and GLA and inhibited formation of the AGE/ALE N(epsilon)-(carboxymethyl)lysine during reaction of GO and GLA with bovine serum albumin. Our data suggest that the AGE/ALE inhibitory activity and the therapeutic effects of PM observed in diabetic animal models depend, at least in part, on its ability to trap reactive carbonyl intermediates in AGE/ALE formation, thereby inhibiting the chemical modification of tissue proteins.

Transaldolase B: Trapping of schiff base intermediate between dihydroxyacetone and ϵ-amino group of active-site lysine residue by borohydride reduction

This chapter describes protocols used for the purification of recombinant transaldolase B from Escherichia coli, the stoichiometric reduction of the Schiff-base intermediate, and subsequent crystallization and structure determination of the enzyme-dihydroxyacetone complex. Trapping of Schiff-base complex is carried out to obtain a crystal structure of this complex that requires a complete conversion of the free enzyme into the enzyme-intermediate complex for subsequent crystallization. This is achieved by two modifications of previous procedures: (1) a high molar excess of fructose 6-phosphate during incubation and borohydride reduction and (2) the enzymatic removal of the product glyceraldehyde 3-phosphate to drive the equilibrium further toward complete complex formation. Catalysis by class I aldolases occurs via a covalent Schiff base intermediate, formed between a catalytic lysine residue and the substrate. In several cases, reduction of this intermediate by borohydride and subsequent peptide sequencing has been used successfully to identify the nature of the amino acid that is covalently modified.

Determination of Active Site Residues in Escherichia coli Endonuclease VIII

Endonuclease VIII from Escherichia coli is a DNA glycosylase/lyase that removes oxidatively damaged bases. EndoVIII is a functional homologue of endonuclease III, but a sequence homologue of formamidopyrimidine-DNA glycosylase (Fpg). Using multiple sequence alignments, we have identified six target residues in endoVIII that may be involved in the enzyme's glycosylase and/or lyase functions: the N-terminal proline, and five acidic residues that are completely conserved in the endoVIII-Fpg proteins. To investigate the contribution of these residues, site-directed mutagenesis was used to create seven mutants: P2T, E3D, E3Q, E6Q, D129N, D160N, and E174Q. Each mutant was assayed both for lyase activity on abasic (AP) sites and for glycosylase/lyase activity on 5-hydroxyuracil, thymine glycol, and gamma-irradiated DNA with multiple lesions. The P2T mutant did not have lyase or glycosylase/lyase activity but could efficiently form Schiff base intermediates on AP sites. E6Q, D129N, and D160N behaved essentially as endoVIII in all assays. E3D, E3Q, and E174Q retained significant AP lyase activity but had severely diminished or abolished glycosylase/lyase activities on the DNA lesions tested. These studies provide detailed predictions concerning the active site of endoVIII.

T4 endonuclease V: Use of NMR and borohydride trapping to provide evidence for covalent enzyme-substrate imine intermediate

The experimental methodologies used in earlier studies on DNA strand breakage characterized the nature of the ends of the product DNA strands, and the experimental data are consistent with the proposed β-elimination mechanism. These interpretations implied a Schiff base (imine) intermediate in the DNA strand breakage reaction and a mechanistic parallel with the aldolase reaction. In this class of enzyme mechanisms, the formation of the protonated imine labilizes the bond from the adjacent carbon to carbon or hydrogen. These studies constituted an early demonstration of the efficacy of amino group reductive methylation coupled with biochemical and NMR experiments in elucidating the involvement of specific residues in enzyme catalysis. The methodology could be more widely applied in the investigation of enzymes utilizing Schiff base intermediates. For example, [ 13 C] formaldehyde-labeled active site amino groups may be used as spectroscopic (NMR) probes to study the structural aspects of substrate binding. Similarly, the base excision repair glycosylases use a base flipping mechanism to achieve access to the scissile base-sugar bond. 13 C-Labeled active site amines may be used as probes of this process.

Detection of intermediates in reactions catalyzed by PLP-dependent enzymes: O-acetylserine sulfhydrylase and serine-glyoxalate aminotransferase

All pyridoxal 5'-phosphate (PLP)-dependent enzymes catalyze their reaction via the initial formation of an external Schiff base intermediate. The internal and external Schiff bases exist in several different tautomeric forms. The chapter illustrates two predominant forms, the enolimine and ketoenamine tautomers, along with the ranges of their respective λ max values. It shows ketoenamine form in the case of the external Schiff base. The external Schiff base is formed via the intermediacy of geminal-diamine intermediates. Once the external Schiff base is formed, the reaction pathway is determined by the kind of reaction catalyzed, and this is determined by the identity of the functional group on the a-carbon that is orthogonal to the PLP ring. O-acetylserine sulfhydrylase (OASS) catalyzes the final step in the de novo synthesis of L-cysteine in enteric bacteria, that is, the conversion of O-acetyl-L-serine (OAS) and bisulfide to L-cysteine and acetate. The cysK gene, encoding for OASS-A, has been cloned and sequenced from Salmonella typhimurium , and the enzyme overexpressed using the wild-type cys K promoter.

SYNTHESIS AND CHARACTERIZATION OF SOME METAL COMPLEXES OF A SCHIFF BASE DERIVED FROM NINHYDRIN AND α ,L-ALANINE

Complexes of Mn(II), Fe(III), Co(II), Ni(II) and Zn(II) with an intermediate Schiff base derived from ninhydrin and a ,L-alanine (indane-1,3-dione-2-imine-N-2-propionate, IDIP) were successfully synthesized. All complexes were distinctly colored and were characterized by elemental analysis, molar conductance, magnetic susceptibility, infrared and electronic spectral studies. The ligand (Schiff base) was shown to behave as a monobasic tridentate ONO donor. The Mn(II) and Fe(III) complexes contain only one ligand molecule plus water and chloride(s) per metal ion, while all the others contain two ligand molecules per metal ion. An octahedral geometry is proposed for the metal complexes. A comparative anti-microbial study of the complexes was undertaken against two gram-negative bacteria, Escherichia coli and Proteus mirabilis and one gram-positive bacterium Staphylococcus aureus. It was revealed that most of the complexes and particularly Mn(II), Fe(III) and Co(II) complexes, have a much enhanced activity relative to ninhydrin against Proteus mirabilis and Staphylococcus aureus. All complexes showed a reduced activity against Escherichia coli. (Received January 4, 2002; revised March 4, 2002) Bull. Chem. Soc. Ethiop. 2002, 16(1), 53-64

Observation of covalent intermediates in an enzyme mechanism at atomic resolution.

In classical enzymology, intermediates and transition states in a catalytic mechanism are usually inferred from a series of biochemical experiments. Here, we derive an enzyme mechanism from true atomic-resolution x-ray structures of reaction intermediates. Two ultra-high resolution structures of wild-type and mutant d-2-deoxyribose-5-phosphate (DRP) aldolase complexes with DRP at 1.05 and 1.10 angstroms unambiguously identify the postulated covalent carbinolamine and Schiff base intermediates in the aldolase mechanism. In combination with site-directed mutagenesis and (1)H nuclear magnetic resonance, we can now propose how the heretofore elusive C-2 proton abstraction step and the overall stereochemical course are accomplished. A proton relay system appears to activate a conserved active-site water that functions as the critical mediator for proton transfer.

Preparation of polythioamides from dialdehydes and diamines with sulfur by the Willgerodt–Kindler type reaction

AbstractWillgerodt–Kindler type reactions of dialdehydes and diamines in the presence of sulfur were investigated for preparation of polythioamides. The one‐pot, three‐component polycondensation afforded various polythioamides in moderate to good yields. The appropriate reaction conditions were examined for the separate monomers. The results led us to the proposed mechanism for the polycondensation including formation of the intermediate Schiff base polymers followed by the successive nucleophilic attack of polysulfide anions to the azomethine units to give the thioamide groups. Structure, solubility, and thermal properties of the polythioamides were also compared with those of analogous polyamides. © 2001 John Wiley & Sons, Inc. J Polym Sci Part A: Polym Chem 39: 3739–3750, 2001

Characterization and mutagenesis of the recombinant N-acetylneuraminate lyase from Clostridium perfringens: insights into the reaction mechanism.

The N-acetylneuraminate lyase from Clostridium perfringens was expressed in Escherichia coli as a fusion protein with a His-tag and purified to homogeneity using metal chelate affinity and anion exchange chromatography. The purified enzyme has a pH optimum of 7.6 and a temperature optimum of 65-70 degrees C. In kinetic studies the lyase exhibits a Km of 3.2 mM for Neu5Ac and a Vmax of 27.5 U x mg(-1). To clarify the functional role of some putative active site residues, site-directed mutagenesis was performed. Lysine 161 was identified as the residue forming the Schiff base intermediate with the substrate. Tyrosine 133 was shown to be also a catalytically important residue; it seems to function as an acceptor for the proton of the C4 hydroxyl group, as already suggested by other groups. Furthermore, it is involved in stabilizing the Schiff base intermediate. Mutations of aspartate 187 and glutamate 188 indicate that both residues are involved in substrate binding. In this respect the carboxy group of aspartate 187 seems to be particularly important. Based on the results of these studies, a model of the reaction mechanism is discussed.

Repair activities of 8-oxoguanine DNA glycosylase from Archaeoglobus fulgidus, a hyperthermophilic archaeon

Oxidative DNA damage is caused by reactive oxygen species formed in cells as by products of aerobic metabolism or of oxidative stress. The 8-oxoguanine (8-oxoG) DNA glycosylase from Archaeoglobus fulgidus (Afogg), which excises an oxidatively-damaged form of guanine, was overproduced in Escherichia coli, purified and characterized. A. fulgidus is a sulfate-reducing archaeon, which grows at between 60 and 95°C, with an optimum growth at 83°C. The Afogg enzyme has both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activities, with the latter proceeding through a Schiff base intermediate. As expected for a protein from a hyperthermophilic organism, the enzyme activity is optimal near pH 8.5 and 60°C, denaturing at 80°C, and is thermally stable at high levels of salt (500mM). The Afogg protein efficiently cleaves oligomers containing 8-oxoG:C and 8-oxoG:G base pairs, and is less effective on oligomers containing 8-oxoG:T and 8-oxoG:A mispairs. While the catalytic action mechanism of Afogg protein is likely similar to the human Ogg1 (hOgg1), the DNA recognition mechanism and the basis for 8-oxoG substrate specificity of Afogg differ from that of hOgg.

Synthesis and antibacterial activities of quaternary ammonium salt of chitosan

Chitosan derivatives with quaternary ammonium salt, such as N, N, N-trimethyl chitosan, N– N-propyl- N, N-dimethyl chitosan and N-furfuryl- N, N-dimethyl chitosan were prepared using different 96% deacetylated chitosan of M v 2.14×10 5, 1.9×10 4, 7.8×10 3. Amino groups on chitosan react with aldehydes to from a Schiff base intermediate. Quaternized chitosan were obtained by reaction of a Schiff base with methyl iodide. The yields, degree of quaternization and water-solubility of quaternized chitosan were influenced by the molecular weight of the chitosan sample. The antibacterial activities of quaternized chitosan against Escherichia coli were explored by calculation of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in water, 0.25 and 0.50% acetic acid medium. Results show the antibacterial activities of quaternized chitosan against E. coli is related to its molecular weight. Antibacterial activities of quaternized chitosan in acetic acid medium is stronger than that in water. Their antibacterial activities is increased as the concentration of acetic acid is increased. It was also found that the antibacterial activity of quaternized chitosan against E. coli is stronger than that of chitosan.

Covalent intermediate trapped in 2-keto-3-deoxy-6- phosphogluconate (KDPG) aldolase structure at 1.95-A resolution.

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyzes the reversible cleavage of KDPG to pyruvate and glyceraldehyde-3-phosphate. The enzyme is a class I aldolase whose reaction mechanism involves formation of Schiff base intermediates between Lys-133 and a keto substrate. A covalent adduct was trapped by flash freezing KDPG aldolase crystals soaked with 10 mM pyruvate in acidic conditions at pH 4.6. Structure determination to 1.95-A resolution showed that pyruvate had undergone nucleophilic attack with Lys-133, forming a protonated carbinolamine intermediate, a functional Schiff base precursor, which was stabilized by hydrogen bonding with active site residues. Carbinolamine interaction with Glu-45 indicates general base catalysis of several rate steps. Stereospecific addition is ensured by aromatic interaction of Phe-135 with the pyruvate methyl group. In the native structure, Lys-133 donates all of its hydrogen bonds, indicating the presence of an epsilon-ammonium salt group. Nucleophilic activation is postulated to occur by proton transfer in the monoprotonated zwitterionic pair (Glu-45/Lys-133). Formation of the zwitterionic pair requires prior side chain rearrangement by protonated Lys-133 to displace a water molecule, hydrogen bonded to the zwitterionic residues.

Production, characterization, and application of anti-gossypol polyclonal antibodies.

This paper describes the development of antibodies for detection of gossypol in cottonseed. Gossypol was linked to LPH (Limulus polyphemus hemolymph), BSA (bovine serum albumin), amines, or amino acids via Schiff base intermediates for the production of immunogen, solid-phase, and gossypol derivatives. Products were stabilized using NaBH(3)CN. Polyclonal antibodies were produced from rabbits after four immunizations with gossypol-LPH. In ELISA, approximately 0.4 mu caused 50% inhibition of binding (I(50)) to gossypol-BSA solid-phase, when gossypol derivatives were used competitively. This antibody showed higher affinity to lysine-derivatized gossypol, hemigossypol, gossypolone, 6, 6'-dimethoxygossypol, and hexamethoxygossypol compared to underivatized counterparts, and this antibody did not recognize naphthalene or naphthol. The ELISA results for acetone-extractable gossypol in cottonseed products (derivatized with L-(+)-lysine) showed good correlation (r(2) = 0.96) with free gossypol results obtained using the official AOCS method.

Ascorbate regeneration by the reduced form of 2-amino-3-carboxy-1, 4-naphthoquinone, a strong growth stimulator for bifidobacteria.

Nonenzymatic reduction of dehydroascorbate into ascorbate by the reduced form (quinol form) of 2-amino-3-carboxy-1,4-naphthoquinone, a strong growth stimulator for bifidobacteria, has been found. The bimolecular reaction rate constant was evaluated as 9 M(-)(1) s(-)(1) at pH 7.0. This reaction has been successfully coupled with enzymatic regeneration of the naphthoquinol by NAD(P)H in cell-free extracts of Bifidobacterium longum 6001. The overall reaction is a regeneration of NAD(P)(+) by dehydroascorbate [or a regeneration of ascorbate by NAD(P)H], in which the naphthoquinone/quinol redox couple functions as an electron transfer mediator. Kinetic study of the reduction of dehydroascorbate with related quinol compounds suggested the significance of the amino substituent of the naphthoquinol. A mechanism of the electron transfer from the quinol to dehydroascorbate is proposed, where the first step of the reaction is a nucleophilic addition of the C(2)-amino substituent of the naphthoquinol to the C(2)-position of dehydroascorbate to form a Schiff base intermediate.

Recognition of Formamidopyrimidine by Escherichia coli and Mammalian Thymine Glycol Glycosylases: DISTINCTIVE PAIRED BASE EFFECTS AND BIOLOGICAL AND MECHANISTIC IMPLICATIONS

The activity of prokaryotic and mammalian thymine glycol (Tg) glycosylases including Escherichia coli endonuclease III (Endo III) and endonuclease VIII (Endo VIII) and mouse Endo III homologue (mNth1) for formamidopyrimidine (Fapy) has been investigated using defined oligonucleotide substrates. 2, 6-Diamino-4-hydroxy-5-N-methylformamidopyrimidine, a methylated Fapy derived from guanine, was site specifically incorporated in the oligonucleotide. The substrates containing Fapy:N pairs (N = A, G, C, T) as well as a Tg:A pair, a physiological substrate of Endo III, Endo VIII, and mNth1, were treated by the enzymes and nicked products were quantified by gel electrophoresis. The activity of Endo III and Endo VIII for Fapy varied markedly depending on the paired base, being the highest with G (activity relative to Tg = 0. 55 (Endo III) and 0.41 (Endo VIII)) and the lowest with C (0.05 (Endo III) and 0.06 (Endo VIII)). In contrast, mNth1 recognized all Fapy pairs equally well and the activity was comparable to Tg. The results obtained in the nicking assay were further substantiated by the analysis of the Schiff base intermediate using NaBH(4) trapping assays. These results indicate that Escherichia coli and mammalian Tg glycosylases have a potential activity to recognize Fapy. However, as demonstrated for Fapy:C pairs, their distinctive activities implicate unequal participation in the repair of Fapy lesions in cells.