Abstract:Pyruvate formate-lyase (acetyl-CoA:formate C-acetyltransferase, EC 2.3.1.54) from anaerobic Escherchia coli cells converts pyruvate to acetyl-CoA and formate by a unique homolytic mechanism that involves a free radical harbored in the protein structure. By EPR spectroscopy of selectively 13C-labeled enzyme, the radical (g = 2.0037) has been assigned to carbon-2 of a glycine residue. Estimated hyperfine coupling constants to the central 13C nucleus (Au = 4.9 mT and A, = 0.1 mT) and to 13C nuclei in a and 13 pos… Show more
“…Reversal may involve loss of O # from ROOd, a relatively facile process when the resultant carboncentred species is stabilized (as it will be here, since backbone α-carbon radicals are capto-dative species stabilized by both the carbonyl and amide functions). This glycyl α-carbon radical occurs in the sequence -Ser-Gly-Tyr-which is cleaved with formation of an N-terminal oxalyl residue from the glycine [11], consistent with the α-amidation fragmentation mechanism [10].…”
Section: Damage By Endogenous Radicalssupporting
confidence: 60%
“…This general scheme has been confirmed for many amino acids, including aromatic amino acids, although other reactions may also occur. Notable quantitative studies were contributed by Garrison, who also proposed a ' peptide α-amidation ' scheme for the oxidative breakage of polypeptide backbones ( [8,9] ; reviewed in [10]), which has been confirmed in some cases [11], and seems to be a common component of chain-fragmentation reactions.…”
Section: Table 1 New Moieties Generated By Biological Protein Oxidatimentioning
Radical-mediated damage to proteins may be initiated by electron leakage, metal-ion-dependent reactions and autoxidation of lipids and sugars. The consequent protein oxidation is O2-dependent, and involves several propagating radicals, notably alkoxyl radicals. Its products include several categories of reactive species, and a range of stable products whose chemistry is currently being elucidated. Among the reactive products, protein hydroperoxides can generate further radical fluxes on reaction with transition-metal ions; protein-bound reductants (notably dopa) can reduce transition-metal ions and thereby facilitate their reaction with hydroperoxides; and aldehydes may participate in Schiff-base formation and other reactions. Cells can detoxify some of the reactive species, e.g. by reducing protein hydroperoxides to unreactive hydroxides. Oxidized proteins are often functionally inactive and their unfolding is associated with enhanced susceptibility to proteinases. Thus cells can generally remove oxidized proteins by proteolysis. However, certain oxidized proteins are poorly handled by cells, and together with possible alterations in the rate of production of oxidized proteins, this may contribute to the observed accumulation and damaging actions of oxidized proteins during aging and in pathologies such as diabetes, atherosclerosis and neurodegenerative diseases. Protein oxidation may also sometimes play controlling roles in cellular remodelling and cell growth. Proteins are also key targets in defensive cytolysis and in inflammatory self-damage. The possibility of selective protection against protein oxidation (antioxidation) is raised.
“…Reversal may involve loss of O # from ROOd, a relatively facile process when the resultant carboncentred species is stabilized (as it will be here, since backbone α-carbon radicals are capto-dative species stabilized by both the carbonyl and amide functions). This glycyl α-carbon radical occurs in the sequence -Ser-Gly-Tyr-which is cleaved with formation of an N-terminal oxalyl residue from the glycine [11], consistent with the α-amidation fragmentation mechanism [10].…”
Section: Damage By Endogenous Radicalssupporting
confidence: 60%
“…This general scheme has been confirmed for many amino acids, including aromatic amino acids, although other reactions may also occur. Notable quantitative studies were contributed by Garrison, who also proposed a ' peptide α-amidation ' scheme for the oxidative breakage of polypeptide backbones ( [8,9] ; reviewed in [10]), which has been confirmed in some cases [11], and seems to be a common component of chain-fragmentation reactions.…”
Section: Table 1 New Moieties Generated By Biological Protein Oxidatimentioning
Radical-mediated damage to proteins may be initiated by electron leakage, metal-ion-dependent reactions and autoxidation of lipids and sugars. The consequent protein oxidation is O2-dependent, and involves several propagating radicals, notably alkoxyl radicals. Its products include several categories of reactive species, and a range of stable products whose chemistry is currently being elucidated. Among the reactive products, protein hydroperoxides can generate further radical fluxes on reaction with transition-metal ions; protein-bound reductants (notably dopa) can reduce transition-metal ions and thereby facilitate their reaction with hydroperoxides; and aldehydes may participate in Schiff-base formation and other reactions. Cells can detoxify some of the reactive species, e.g. by reducing protein hydroperoxides to unreactive hydroxides. Oxidized proteins are often functionally inactive and their unfolding is associated with enhanced susceptibility to proteinases. Thus cells can generally remove oxidized proteins by proteolysis. However, certain oxidized proteins are poorly handled by cells, and together with possible alterations in the rate of production of oxidized proteins, this may contribute to the observed accumulation and damaging actions of oxidized proteins during aging and in pathologies such as diabetes, atherosclerosis and neurodegenerative diseases. Protein oxidation may also sometimes play controlling roles in cellular remodelling and cell growth. Proteins are also key targets in defensive cytolysis and in inflammatory self-damage. The possibility of selective protection against protein oxidation (antioxidation) is raised.
“…1A). 7 , 10 The use of protein-based radicals enables these enzymes to perform a diverse set of chemically challenging transformations (Fig. 1A).…”
Section: Introductionmentioning
confidence: 99%
“…6 , 7 , 10 Due to their susceptibility to inactivation by molecular oxygen, both GREs and AEs are only present in facultative and obligate anaerobes. 10 , 11
10.1080/19490976.2018.1435244-F0001Figure 1.(A) General mechanism of glycyl radical enzymes (GREs). A partner radical S -adenosylmethionine (SAM) activating enzyme (AE) first abstracts a hydrogen atom from the α-carbon of a conserved active site glycine residue on the GRE to generate a carbon-centered radical.…”
The discovery of enzymes responsible for previously unappreciated microbial metabolic pathways furthers our understanding of host-microbe and microbe-microbe interactions. We recently identified and characterized a new gut microbial glycyl radical enzyme (GRE) responsible for anaerobic metabolism of trans-4-hydroxy-l-proline (Hyp). Hyp dehydratase (HypD) catalyzes the removal of water from Hyp to generate Δ1-pyrroline-5-carboxylate (P5C). This enzyme is encoded in the genomes of a diverse set of gut anaerobes and is prevalent and abundant in healthy human stool metagenomes. Here, we discuss the roles HypD may play in different microbial metabolic pathways as well as the potential implications of this activity for colonization resistance and pathogenesis within the human gut. Finally, we present evidence of anaerobic Hyp metabolism in sediments through enrichment culturing of Hyp-degrading bacteria, highlighting the wide distribution of this pathway in anoxic environments beyond the human gut.
“…For aromatic amino acid residues, cleavage of the C ␣ -C  bond eliminates pquinomethide from tyrosine and 3-methylene indolenine from tryptophan, yielding a glycyl radical with the unpaired electron located on the ␣-carbon [12,27,28,30]. Such ␣-centered radicals have been identified in some anaerobic enzymes [41][42][43], including pyruvate formatelyase [44,45], anaerobic ribonucleotide reductase [46], benzylsuccinate synthase [47,48], 4-hydroxyphenylacetate decarboxylase [49], and glycerol dehydratase [50]. A radical transfer from the thiyl group in the cysteine residue to a neighboring glycine residue by ␣-hydrogen-atom abstraction generates an ␣-centered radical [51,52], which is stabilized by -electron delocalization between the adjacent electron-withdrawing carbonyl group and the electron-donating amide nitrogen.…”
[7,8], infrared multiphoton dissociation (IRMPD) [9], and blackbody radiation [10]. The mechanisms by which protonated peptides dissociate have, therefore, generated much interest [11,12]. Charge-driven amide bond cleavages are induced by the mobile proton, giving b-or y-type ions, which are, respectively, the N-or C-terminal fragments [1,2]. By contrast, open-shell peptide radical cations produced in electron-capture [13][14][15] or electron-transfer dissociation experiments [16] give c-or z-type ions, by cleaving the N-C ␣ bond adjacent to the aminoketyl radical being formed [17][18][19][20]. The substantial fragmentation differences between protonated and radical cationic peptide provide useful complementary peptide sequencing information [21][22][23]. Ultraviolet (UV) photodissociation has also been employed for peptide fragmentation [24]. UV photodissociation of a peptide/protein chemically modified to incorporate an appropriate chromophore can generate a radical cation that undergoes site-specific, radical-driven dissociations, which are useful in protein identifications [25,26]. Peptide radical cations have also been generated via low-energy CID of a ternary metal complex containing the peptide and auxiliary ligands [27][28][29][30][31][32][33][34][35][36][37]. These methodologies open new avenues whereby the fragmentation chemistries of peptide radical cations can be examined and exploited.Dissociation at an amino-acid side chain is commonplace in the CID of peptide radical cations. These fragmentations are useful in providing differentiating signatures for isobaric residues present [14, 29, 38 -40], e.g., between leucine and isoleucine [14,29], and between aspartic acid and isoaspartic acid [38,39]. For aromatic amino acid residues, cleavage of the C ␣ -C  bond eliminates pquinomethide from tyrosine and 3-methylene indolenine from tryptophan, yielding a glycyl radical with the unpaired electron located on the ␣-carbon [12,27,28,30]. Such ␣-centered radicals have been identified in some anaerobic enzymes [41][42][43] [50]. A radical transfer from the thiyl group in the cysteine residue to a neighboring glycine residue by ␣-hydrogen-atom abstraction generates an ␣-centered radical [51,52], which is stabilized by -electron delocalization between the adjacent electron-withdrawing carbonyl group and the electron-donating amide nitrogen. This stabilization is known as the captodative effect [53]. The intrinsic stability of an amino-acid captodative radical has recently been verified by IRMPD spectroscopy on the histidine radical cation in the Address reprint requests to Professor K
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