Cα-Cβ Bond Cleavage Research Articles

Identification of DypB from Rhodococcus jostii RHA1 as a Lignin Peroxidase

Rhodococcus jostii RHA1, a polychlorinated biphenyl-degrading soil bacterium whose genome has been sequenced, shows lignin degrading activity in two recently developed spectrophotometric assays. Bioinformatic analysis reveals two unannotated peroxidase genes present in the genome of R. jostii RHA1 with sequence similarity to open reading frames in other lignin-degrading microbes. They are members of the Dyp peroxidase family and were annotated as DypA and DypB, on the basis of bioinformatic analysis. Assay of gene deletion mutants using a colorimetric lignin degradation assay reveals that a ΔdypB mutant shows greatly reduced lignin degradation activity, consistent with a role in lignin breakdown. Recombinant DypB protein shows activity in the colorimetric assay and shows Michaelis-Menten kinetic behavior using Kraft lignin as a substrate. DypB is activated by Mn(2+) by 5-23-fold using a range of assay substrates, and breakdown of wheat straw lignocellulose by recombinant DypB is observed over 24-48 h in the presence of 1 mM MnCl(2). Incubation of recombinant DypB with a β-aryl ether lignin model compound shows time-dependent turnover, giving vanillin as a product, indicating that C(α)-C(β) bond cleavage has taken place. This reaction is inhibited by addition of diaphorase, consistent with a radical mechanism for C-C bond cleavage. Stopped-flow kinetic analysis of the DypB-catalyzed reaction shows reaction between the intermediate compound I (397 nm) and either Mn(II) (k(obs) = 2.35 s(-1)) or the β-aryl ether (k(obs) = 3.10 s(-1)), in the latter case also showing a transient at 417 nm, consistent with a compound II intermediate. These results indicate that DypB has a significant role in lignin degradation in R. jostii RHA1, is able to oxidize both polymeric lignin and a lignin model compound, and appears to have both Mn(II) and lignin oxidation sites. This is the first detailed characterization of a recombinant bacterial lignin peroxidase.

Carbon-13 CP/MAS solid state NMR and X-ray diffraction spectroscopy studies on lime wood decayed by Chaetomium globosum

The degradation of the plant cell wall by microorganisms has been studied by X-ray diffraction, and solid state CP/MAS 13C NMR spectroscopy. Lime wood ( Tilia cordata Miller) samples were inoculated with Chaetomium globosum for various durations up to 133 days. Structural modifications were assessed by comparing decayed lime wood samples with reference. These methods proved to be able to give insights into the modifications at a molecular level of the cell wall components by Ascomycetes fungi attack. Significant changes in relative crystallinity and apparent lateral crystallite size, as measured by XRD, were detectable relatively early in the decay process. The content of hemicelluloses and cellulose in the wood samples decreased after biodegradation with Chaetomium globosum. The main chemical changes in wood during decay are the loss of hemicelluloses and cellulose simultaneously with lesser changes in lignin structure, which mainly consist in partial loss of methoxyl groups and Cα–Cβ bond cleavage and loss of β-O-4 linkages.

Reactions of an Aromatic σ,σ-Biradical with Amino Acids and Dipeptides in the Gas Phase

Gas-phase reactivity of a positively charged aromatic σ,σ-biradical (N-methyl-6,8-didehydroquinolinium) was examined toward six aliphatic amino acids and 15 dipeptides by using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR) and laser-induced acoustic desorption (LIAD). While previous studies have revealed that H-atom and NH(2) abstractions dominate the reactions of related monoradicals with aliphatic amino acids and small peptides, several additional, unprecedented reaction pathways were observed for the reactions of the biradical. For amino acids, these are 2H-atom abstraction, H(2)O abstraction, addition - CO(2), addition - HCOOH, and formation of a stable adduct. The biradical reacts with aliphatic dipeptides similarly as with aliphatic amino acids, but undergoes also one additional reaction pathway, addition/C-terminal amino acid elimination (addition - CO - NHCHR(C)). These reactions are initiated by H-atom abstraction by the biradical from the amino acid or peptide, or nucleophilic addition of an NH(2) or a HO group of the amino acid or peptide at the radical site at C-6 in the biradical. Reactions of the unquenched C-8 radical site then yield the products not observed for related monoradicals. The biradical reacts with aromatic dipeptides with an aromatic ring in N-terminus (i.e., Tyr-Leu, Phe-Val, and Phe-Pro) similarly as with aliphatic dipeptides. However, for those aromatic dipeptides that contain an aromatic ring in the C-terminus (i.e., Leu-Tyr and Ala-Phe), one additional pathway, addition/N-terminal amino acid elimination (addition - CO - NHCHR(N)), was observed. This reaction is likely initiated by radical addition of the biradical at the aromatic ring in the C-terminus. Related monoradicals add to aromatic amino acids and small peptides, which is followed by Cα-Cβ bond cleavage, resulting in side-chain abstraction by the radical. For biradicals, with one unquenched radical site after the initial addition, the reaction ultimately results in the loss of the N-terminal amino acid. Similar to monoradicals, the C-S bond in amino acids and dipeptides was found to be especially susceptible to biradical attack.

Catalytic Activity of the Anaerobic Tyrosine Lyase Required for Thiamine Biosynthesis in Escherichia coli

Thiazole synthase in Escherichia coli is an alphabeta heterodimer of ThiG and ThiH. ThiH is a tyrosine lyase that cleaves the C alpha-C beta bond of tyrosine, generating p-cresol as a by-product, to form dehydroglycine. This reactive intermediate acts as one of three substrates for the thiazole cyclization reaction catalyzed by ThiG. ThiH is a radical S-adenosylmethionine (AdoMet) enzyme that utilizes a [4Fe-4S](+) cluster to reductively cleave AdoMet, forming methionine and a 5'-deoxyadenosyl radical. Analysis of the time-dependent formation of the reaction products 5'-deoxyadenosine (DOA) and p-cresol has demonstrated catalytic behavior of the tyrosine lyase. The kinetics of product formation showed a pre-steady state burst phase, and the involvement of DOA in product inhibition was identified by the addition of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase to activity assays. This hydrolyzed the DOA and changed the rate-determining step but, in addition, substantially increased the uncoupled turnover of AdoMet. Addition of glyoxylate and ammonium inhibited the tyrosine cleavage reaction, but the reductive cleavage of AdoMet continued in an uncoupled manner. Tyrosine analogues were incubated with ThiGH, which showed a strong preference for phenolic substrates. 4-Hydroxyphenylpropionic acid analogues allowed uncoupled AdoMet cleavage but did not result in further reaction (C alpha-C beta bond cleavage). The results of the substrate analogue studies and the product inhibition can be explained by a mechanistic hypothesis involving two reaction pathways, a product-forming pathway and a futile cycle.

Mass-spectrometric and computational study of tryptophan radicals (Trp + H)˙ produced by collisional electron transfer to protonated tryptophan in the gas phase

Hydrogen atom adducts to tryptophan were generated for the first time in the gas phase by collisional electron transfer to protonated tryptophan at 7170 eV kinetic energy. The radicals showed fast dissociations by C(α)-C(β) bond cleavage and cross-ring cleavages occurring on the 7.3 μs time scale. The mechanism of the C(α)-C(β) bond cleavage was explained by ab initio computational analysis of the radical potential energy surface. This showed spontaneous isomerization of the primary tryptophan radical by ammonium hydrogen atom migration to the carboxyl group. The stable intermediate formed by the isomerization can undergo radical-induced scission of the C(α)-C(β) bond in competition with H-atom migrations to the C-2 and C-4 positions of the indole ring. RRKM calculations of unimolecular rate constants on the B3-ROMP2/6-311++G(2d,p) potential energy surface indicated that the C(α)-C(β) bond cleavage was the fastest unimolecular reaction of the radical intermediates within the range of internal energies acquired upon electron transfer. We also report an updated G2(MP2) proton affinity of tryptophan (PA = 946 kJ mol(-1)) and hydrogen atom affinities of the tryptophan indole ring of relevance to electron-based peptide dissociations.

Synthesis of a lignin polymer model consisting of only phenolic β-O-4 linkages and testing its reactivity under alkaline conditions 10th EWLP, Stockholm, Sweden, August 25–28, 2008

Abstract A trimeric β-O-4 lignin model bearing a styrene unit was polymerized in the presence of azobisisobutyronitrile as an initiator of free radical polymerization. The polymer analysis achieved by size exclusion chromatography (SEC), 1H, 13C, 31P NMR, matrix-assisted laser desorption ionization combined with time-of-flight mass spectrometry (MALDI-ToF MS), differential scanning calorimetry, and thermogravimetry indicated its well-defined structure with good thermal stability at temperatures usually applied for alkaline pulping. SEC analysis proved a high degree of polymerization despite the size of the phenolic β-O-4 units in the polymer structure. MALDI-ToF spectrometry indicated that the polymer is composed of phenolic β-O-4 elements only. The chemical reactivity under alkaline conditions of the polymer was studied by both HPLC and 31P NMR. These two methods revealed two main concurrent processes, which include an oxidation of the phenol part followed by Cα-Cβ bond cleavage, and a formation of a quinone methide followed by its β-O-4 bond cleavage.

Homolytic CαCβ Bond Cleavage in a Chiral Alkylarene Radical Cation: Effects of Asymmetric Microsolvation

In the Communication “Homolytic CαCβ Bond Cleavage in a Chiral Alkylarene Radical Cation: Effects of Asymmetric Microsolvation" by M. Speranza et al.1 ([DOI: 10.1002/anie.200353462]) on page 1870, second paragraph, the sentences “In particular, the heterochiral complex exhibits shorter OH⋅⋅⋅OH and OH⋅⋅⋅α hydrogen-bond distances than the homochiral adduct. This implies that BDSS is a better H-bond acceptor than BDRR when associated with the chiral (BZC2H5)R moiety.” should be replaced by “In particular, the homochiral complex exhibits a shorter OH⋅⋅⋅OH distance (2.006 Å) than the heterochiral adduct (2.021 Å). This implies that BDRR is a better H-bond acceptor than BDSS when associated with the chiral (BZC2H5)R moiety.” On page 1871, first paragraph, the last sentence “This difference may be attributed to structural factors that make solv in the former adduct a better H-bond acceptor than in the latter, although other hypotheses, such as that involving the interaction of the solvent molecule with the Cα center of [(BZC2H5)R]+ cannot be excluded.” should be replaced by “This difference may be attributed to structural factors that make solv in the latter adduct a better H-bond acceptor than in the former, although other hypotheses, such as that involving the interaction of the solvent molecule with the Cα center of [(BZC2H5)R]+ cannot be excluded.” In the Table of Contents, the sentence “The difference is ascribed to structural factors that make BDSS a better H-bond acceptor than BDRR in their adducts with [(BZC2H5)R]+.” should be replaced by “The difference is ascribed to structural factors that make BDRR a better H-bond acceptor than BDSS in their adducts with [(BZC2H5)R]+.”

Homolytic Calpha--Cbeta bond cleavage in a chiral alkylarene radical cation: effects of asymmetric microsolvation.

Fragmentation of the (R)-(+)-1-phenyl-1-propanol radical cation ([(BZC2H5)R]+) is markedly affected by asymmetric microsolvation (see picture). The CαCβ bond cleavage in the heterochiral [(BZC2H5)R.BDSS]+ cluster is more efficient than in the homochiral [(BZC2H5)R.BDRR]+. The difference is ascribed to structural factors that make BDSS a better H-bond acceptor than BDRR in their adducts with [(BZC2H5)R]+. BZ=PhCHOH, BD=2,3-butanediol.

Photo-induced dissociation of protonated tryptophan TrpH+: A direct dissociation channel in the excited states controls the hydrogen atom loss

Protonated tryptophan ions (TrpH+) are generated by electrospray ionization and dissociated by irradiation with a UV laser. Different photo-fragments are observed among which a new photo-induced dissociation channel leading to the loss of a hydrogen atom that is not observed in conventional collision-induced dissociation. A tryptophan radical cation (Trp+) is produced in this process that subsequently leads to the m/z = 130 fragment through a Cα–Cβ bond cleavage, a typical fragmentation product of the Trp+ radical cation generated either by electron impact or by photo-ionization. These results can be understood considering the excited states of protonated tryptophan: UV excitation of TrpH+ produces a mixed ππ*/πσ* state, the ππ* state being mainly located on the indole chromophore while the πσ* is mainly on the protonated terminal amino group. This πσ* state is repulsive along the N–H bond coordinate and leads either to hydrogen atom detachment producing a Trp+ radical cation that undergoes further fragmentations or to internal conversion to the ground state of the protonated TrpH+ ion.

Selective Cα–Cβ bond cleavage by water in allenylidene and alkenylvinylidene ruthenium complexes

The Ru(II) complex mer,trans-[(PNP)RuCl2(PPh3)] [PNP = MeCH2CH2N(CH2CH2PPh2)2] reacts in refluxing THF with propargyl alcohols HCCCRR′OH (R = R′ = Me, Ph; R = Me, R′ = Ph) yielding either the allenylidene complex fac,cis-[(PNP)RuCl2{CCCPh2}] or the alkenylvinylidene derivatives fac,cis-[(PNP)RuCl2{CC(H)C(R)CH2}] (R = Me, Ph); treatment of all these complexes in CH2Cl2 or THF with water results in the formation of the ruthenium carbonyl fac,cis-[(PNP)RuCl2(CO)] and free alkenes H2CCRR′ (R = R′ = Me, Ph; R = Me, R′ = Ph) via regioselective Cα–Cβ bond cleavage.

Reactions of Metal Ion Complexes with Lignin Model Compounds, Part II. Fe(TSPC) Catalyzed Formation of Oxidized Products in the Absence of Oxygen

Abstract The water soluble phthalocyanine complex trisodium tetra-4-sulfonatophthalocyanineiron(III) (Fe(TSPc)) was found to be an effective catalyst for the cleavage of the β-ether bonds in the phenolic lignin model compounds guaiacylglycol β-guaiacyl ether (1) and guaiacylglycerol β-guaiacyl ether (11). The products of these reactions were very different from those formed in the corresponding reactions catalyzed by anthraquinone (AQ) or Co(SPP).1–4 In particular, they gave large quantities of oxidized products, even though the reactions were performed in the absence of oxygen or other added oxidant. Mechanisms have been proposed for the oxidation reactions involving 1 and 11. In both cases the first step involves one electron oxidation of the lignin model compound by the catalyst. The radical derived from 1 then undergoes further one electron oxidation and deprotonation to give 4′-hydroxy-3′-methoxy-l-(2″-methoxyphenoxy)acetophenone (8) whereas that derived from 11 undergoes Cα-Cβ bond cleavage to give ...

Kinetic Analysis of the Noncompetitive Inhibition of the Lignin-Peroxidase-catalyzed Reaction by Oxalic Acid

Oxalic acid was found to inhibit noncompetitively the Cα-Cβ bond cleavage of veratrylglycerol catalyzed by a lignin peroxidase (LiP) isozyme of the white-rot fungus P. chrysosporium. With greater amounts of oxalic acid in the LiP system, the substrate was not converted to veratraldehyde but was almost all recovered. Oxalic acid was shown to be decomposed to CO2 during the enzymatic reaction. The results clearly indicate that oxalic acid reduced the cation radical intermediate formed in the reaction back to the substrate to block the production of veratraldehyde. A novel equation has been derived to explain the mechanism for this unique non-competitive inhibition that is different from the classical noncompetitive one. The inhibition constant Ki obtained here, which is different from the classical inhibition constant Ki, is defined as the ratio of the rate constant (kp) for product formation to the rate constant (ki) for the reduction of the cation radical to the substrate.

Metabolism of non-phenolic ?-O-4 lignin model compounds by the white-rot fungus Phlebia radiata

The degradation of three non-phenolic β-O-4 diarylpropane lignin model compounds was studied in cultures of the white-rot fungus Phlebia radiata. The degradation pattern of the model compound 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (I) was also compared with that of Phanerochaete chrysosporium under conditions where both fungi were cultivated without agitation in an oxygen atmosphere. Compound I was readily degraded by both fungi, and qualitatively the degradation patterns were quite similar. The product, after Cα-Cβ bond cleavage, was veratraldehyde (IV) which was almost stoichiometrically reduced to veratryl alcohol (V). However, large amounts of V were detected only in P. chrysosporium cultures. Experiments with the model compound 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (II) showed that in the presence of II, the total amount of veratryl compounds accounted for 15–33 μm in standing cultures of Phlebia radiata. The model compound 1-(3,4-dimethoxyphenyl)-2-(4-methoxyphenoxy) propane-1,3-diol (III) was more readily degraded than I and II. The results indicated that, in P. radiata cultures, the acting enzymes were lignin peroxidases and IV reducing enzyme, while laccase was less important.

ChemInform Abstract: Photochemical Oxidative Cα‐Cβ Bond Cleavage of Tryptophan Side‐Chain by Pyrimido[5,4‐g]pteridine N‐Oxide.

Chemischer InformationsdienstVolume 17, Issue 50 Natural Products ChemInform Abstract: Photochemical Oxidative Cα-Cβ Bond Cleavage of Tryptophan Side-Chain by Pyrimido[5,4-g]pteridine N-Oxide. M. SAKO, M. SAKOSearch for more papers by this authorK. SHIMADA, K. SHIMADASearch for more papers by this authorK. HIROTA, K. HIROTASearch for more papers by this authorY. MAKI, Y. MAKISearch for more papers by this author M. SAKO, M. SAKOSearch for more papers by this authorK. SHIMADA, K. SHIMADASearch for more papers by this authorK. HIROTA, K. HIROTASearch for more papers by this authorY. MAKI, Y. MAKISearch for more papers by this author First published: December 16, 1986 https://doi.org/10.1002/chin.198650323Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume17, Issue50December 16, 1986 RelatedInformation

Theoretical study on electron impact mass spectrometry. III ab initio MO study on the fragmentation of propylamine.

In order to elucidate the fragmentation mechanism of propylamine following the low energy electron bombardment, the potential energy curves have been calculated by means of ab initio MO methods (STO-3G/4-31G). The present study indicates that the fragmentation due to Cα-Cβ bond cleavage is the main process at the low energy.

Single-electron transfer processes and the reaction mechanism of enzymic degradation of lignin

The lignin-degrading, ‘H 2O 2-dependent oxygenase’ from Phanerochaete chrysosporium oxidised veratryl alcohol without incorporating oxygen into the substrate. It also catalysed α-β cleavage of a non-phenolic diarylpropane substrate in the absence of oxygen. Studies with tris(phenanthroline)iron(III) established the importance of single-electron transfer in bringing about α-β cleavage. We propose that the lignin-degrading enzyme functions not as an oxygenase but as a peroxidase and that the oxidation reactions are brought about by initial single-electron transfer between the aromatic ring and a high redox oxy-ferryl active site in the enzyme. Lignin degradation Single-electron transfer Cα-Cβ bond cleavage Phanerochaete chrysosporium Peroxidase compound I

Incorporation of dioxygen into the hydroxylated product during the C–C single bond cleavage of 1,2-bis(p-methoxyphenyl)propane-1,3-diol catalysed by hemin. A novel model sysystem for the hemoprotein ligninase

Oxidation of the lignin model compound 1,2-bis(p-methoxyphenyl)propane-1,3-diol (1) catalysed by hemin in the presence of t-butyl hydroperoxide and 18O2 yielded p-methoxyphenylethane-1,2-diol (2) with 83%18O incorporation into the newly formed hydroxy group and p-anisaldehyde (3) as the initial Cα–Cβ bond cleavage products.

Kinetic studies of secondary alcohol photo-oxidation on ZnO and TiO2 at 348 K studied by gas-chromatographic analysis

Kinetics of conversion of propan-2-ol and butan-2-ol to the corresponding ketones by photoassisted dehydrogenation, (–H2)*, to lower aldehydes by a photoassisted Cα—Cβ bond cleavage process, (α–β)*, and to trace quantities of alkene by photoassisted dehydration (–H2O)* have been investigated at oxygen partial pressures in the range 0–700 Torr and at alcohol partial pressures of 0–60 Torr, utilising dynamic flow photoreactors and gas-chromatographic analysis. Qualitatively similar results were obtained either with a ‘continuous reactant flow plus continuous u.v. illumination’ procedure allied to intermittent sampling, or with ‘pulsed reactant and analysis’ procedure. Both procedures yielded dependence upon alcohol pressure indicative of two parallel photoassisted pathways to (–H2)* product, one being Langmuir–Hinshelwood (LH*) and the other Eley–Rideal (ER*) in character. A decline in activity of the metal oxide surfaces as photoassisted conversion of alcohol increased was observed with both procedures and affected the LH* process for (–H2)* more strongly. It is proposed that this LH* process involved alcohol chemisorption and hole localisation at coordinatively unsaturated O2–cus ions. The ER* process is envisaged to have involved encounter of alcohol (from the gas phase or from a reversibly adsorbed weakly bound state) with a much more numerous type of surface location capable of being photoactivated by hole capture. The proposed mechanisms account for observed dependences of the (–H2)* process upon square root of the incident light intensity, upon alcohol pressure and upon oxygen pressure.

Reaktionen von Bleitetraacetat mit alicyclischen Alkoholen, III. Cyclopropyl‐ und Cyclobutylcarbinole. 18. Mitteilung über Reaktionen mit Bleitetraacetat

AbstractWhen treated with lead tetraacetate, cyclopropylcarbinol and cyclopropylmethylcarbinol do not give β‐fragmentation products resulting from the intermediate formation of cyclopropyl radicals; however, cyclopropylmethylcarbinol affords a small amount of a fragmentation product which arises from CαCβ bond cleavage involving removal of a methyl radical. In contrast, cyclobutylcarbinol undergoes β‐fragmentation in 18% yield with formation of both the unrearranged cyclobutyl acetate and the rearranged cyclopropylmethyl acetate. These results suggest the following order of increasing radical stability: cyclopropyl < methyl < cyclobutyl, whereas the isolation of the isomeric fragmentation acetates in the lead tetraacetate reaction of cyclobutylcarbinol represents further evidence that in the β‐fragmentation process the initially produced carbon radical fragment is in major part oxidized to the corresponding carbonium ion before final product formation.