Protein Thiocarboxylate-Dependent Methionine Biosynthesis in Wolinella succinogenes

Thiocarboxylated proteins are important intermediates in a variety of biochemical sulfide transfer reactions. Here we identify a protein thiocarboxylate-dependent methionine biosynthetic pathway in Wolinella succinogenes. In this pathway, the carboxy terminal alanine of a novel sulfur transfer protein, HcyS-Ala, is removed in a reaction catalyzed by a metalloprotease, HcyD. HcyF, an ATP-utilizing enzyme, catalyzes the adenylation of HcyS. HcyS acyl-adenylate then undergoes nucleophilic substitution by bisulfide produced by Sir to give the HcyS thiocarboxylate. This adds to O-acetylhomoserine to give HcyS-homocysteine in a PLP-dependent reaction catalyzed by MetY. HcyD-mediated hydrolysis liberates homocysteine. A final methylation completes the biosynthesis. The biosynthetic gene cluster also encodes the enzymes involved in the conversion of sulfate to sulfide suggesting that sulfate is the sulfur source for protein thiocarboxylate formation in this system.     

Biosynthesis of thiamin thiazole in eukaryotes: conversion of NAD to an advanced intermediate

Thiazole synthase catalyzes the formation of the thiazole moiety of thiamin pyrophosphate. The enzyme from Saccharomyces cerevisiae (THI4) copurifies with a set of strongly bound adenylated metabolites. One of them has been characterized as the ADP adduct of 5-(2-hydroxyethyl)-4-methylthiazole-2-carboxylic acid. Attempts toward yielding active wild-type THI4 by releasing protein-bound metabolites have failed so far. Here, we describe the identification and characterization of two partially active mutants (C204A and H200N) of THI4. Both mutants catalyzed the release of the nicotinamide moiety from NAD to produce ADP-ribose, which was further converted to ADP-ribulose. In the presence of glycine, both the mutants catalyzed the formation of an advanced intermediate. The intermediate was trapped with ortho-phenylenediamine, yielding a stable quinoxaline derivative, which was characterized by NMR spectroscopy and ESI-MS. These observations confirm NAD as the substrate for THI4 and elucidate the early steps of this unique biosynthesis of the thiazole moiety of thiamin in eukaryotes.     

Paracetamol oxidation: synthesis and reactivity of N-acetyl-p-benzoquinoneimine

A convenient synthesis of N-acetyl-p-benzoquinoneimine is described, its reaction with glutathione and N-acetyl cysteine proceeds not only by adduct formation but also by a reductive pathway.     

Total Synthesis of the Nonribosomal Peptide Surugamide B and Identification of a New Offloading Cyclase Family

The cathepsin B inhibitor surugamide B ( 2 ), along with structurally related derivatives (A and C–E), has previously been isolated from the marine actinomycete Streptomyces sp. JAMM992. The biosynthetic genes are unexpectedly part of a cluster of four non‐ribosomal peptide synthetase (NRPS) genes, two of which are responsible for the biosynthesis of the additional linear decapeptide surugamide F. However, the thioesterase domain required for the later stage of the biosynthesis of the cyclic peptides surugamides A–E is not present in any module architecture of the surugamide NRPSs. Herein, we report the first total synthesis of surugamide B ( 2 ) through the macrocyclization at the biomimetic position, which not only alleviated the Cα epimerization in the macrolactamization process, but also efficiently provided 2 in 34 % yield for 18 steps. Furthermore, both the chemical and enzymatic studies with the biosynthetic precursor mimics revealed that the stand‐alone enzyme SurE, which belongs to the penicillin‐binding protein family, is responsible for macrocyclization of the tethered octapeptidyl intermediate.     

Studies on the synthesis and biosynthesis of the fungal alkaloid necatorone

An alternative pathway for the biosynthesis of the fungal alkaloid necatorone has been studied using fluorine-labeled 3-(2-carboxyphenylamino)-l-tyrosine. Although no incorporation of this compound could be detected in feeding experiments with young specimens of Lactarius necator, an analogous 3-aminotyrosine derivative could be converted synthetically into the oxopyridoacridine core structure of necatorone. In experiments aimed at the synthesis of aaptamine-type alkaloids, an unprecedented cyclization of a 3-aminotyrosine-methyl propiolate adduct to a methyl isoquinoline-3-carboxylate was observed. A mechanism is proposed, in which C3 of the propiolate delivers C1 of the isoquinoline nucleus.     

Photochemically catalyzed reaction of ochratoxin A with D- and L-cysteine

The photolysis (>300 nm) of ochratoxin A (OTA, N-[[(3R)-5-chloro-8-hydroxy-3-methyl-1-oxo-7-isochromanyl]carbonyl]-3-phenyl-L-alanine, 1) in the presence of excess (2 and 12 molar equiv) cysteine (CySH) has been investigated and found to yield sulfur adducts 5 and 6 that are characterized by liquid chromatography-mass spectrometry and 1H-NMR spectroscopy. The adduct 5 was ascribed to the Michael addition conjugate resulting from covalent attachment of CySH to the ochratoxin quinone (4) generated by photooxidation of OTA. This species was also formed by photolysis of a synthetic sample of the hydroquinone of OTA (ochratoxin hydroquinone, 3) in the presence of 12 equiv L-CySH. The conjugate 5 derived from photolysis of 3 with L-CySH was used for 1H-NMR analysis. The sulfur adduct 6 was the major species detected from covalent attachment of CySH to photoactivated OTA, and it resulted from direct displacement of the OTA Cl atom by CySH. The implications of the cysteinyl adducts to the in vivo toxicity of OTA are discussed, with particular emphasis given to conjugate 5, as products from the photooxidative pathway may be of relevance to the nephrotoxic properties of OTA.     

Redox-dependent formation of disulfide bonds in human replication protein A

Human replication protein A (RPA) is a single-stranded DNA (ssDNA)-binding protein with three subunits. The largest subunit, p70, contains a conserved (cysteine)(4)-type zinc-finger motif that has been implicated in the regulation of DNA replication and repair. Previous studies indicated that the ssDNA-binding activity of RPA could be redox-regulated via reversible oxidation of cysteines in the zinc-finger motif. We exposed recombinant human RPA to hydrogen peroxide and characterized the oxidized protein by liquid chromatography/tandem mass spectrometric (LC/MS/MS) analyses. Our results demonstrated that, upon H(2)O(2) treatment, four cysteines, which reside at the zinc-finger motif of the p70 subunit, could result in the formation of two pairs of intramolecular disulfides, Cys481-Cys486 and Cys500-Cys503; no cysteine sulfinic acid or cysteine sulfonic acid could be found. Moreover, the other 11 cysteines in this protein remained intact. The results demonstrated that the formation of disulfide bonds at the zinc-finger site was responsible for the redox regulation of the DNA-binding activity of RPA.     

An Aromatic Dyad Motif in Dye Decolourising Peroxidases Has Implications for Free Radical Formation and Catalysis

Dye decolouring peroxidases (DyPs) are the most recent class of heme peroxidase to be discovered. On reacting with H₂O₂, DyPs form a high-valent iron(IV)-oxo species and a porphyrin radical (Compound I) followed by stepwise oxidation of an organic substrate. In the absence of substrate, the ferryl species decays to form transient protein-bound radicals on redox active amino acids. Identification of radical sites in DyPs has implications for their oxidative mechanism with substrate. Using a DyP from Streptomyces lividans, referred to as DtpA, which displays low reactivity towards synthetic dyes, activation with H₂O₂ was explored. A Compound I EPR spectrum was detected, which in the absence of substrate decays to a protein-bound radical EPR signal. Using a newly developed version of the Tyrosyl Radical Spectra Simulation Algorithm, the radical EPR signal was shown to arise from a pristine tyrosyl radical and not a mixed Trp/Tyr radical that has been widely reported in DyP members exhibiting high activity with synthetic dyes. The radical site was identified as Tyr374, with kinetic studies inferring that although Tyr374 is not on the electron-transfer pathway from the dye RB19, its replacement with a Phe does severely compromise activity with other organic substrates. These findings hint at the possibility that alternative electron-transfer pathways for substrate oxidation are operative within the DyP family. In this context, a role for a highly conserved aromatic dyad motif is discussed.     

Top-down approach of completely sequencing a 4.9 kDa recombinant peptide using quadrupole time-of-flight mass spectrometry

A recombinant peptide (near the C-terminal region of head involution defective protein) of 4.9 kDa has been completely sequenced and characterized using medium-resolution mass spectrometry (QTOF). The observed difference in the experimental mass and the theoretical mass is due to beta-mercaptoethanol adduct formation on the cysteine residue. The fragmentation pattern is correlated with the primary structure of the protein. Top-down sequencing of the peptide was extended to small proteins like barstar of mass 10.3 kDa.     

Electrochemical detection of thiols in biological media

The utilisation of catechol as an electrochemical indicator for the presence of sulphydryl thiols (RSH) has been investigated. The electrochemical oxidation of the catechol within tissue culture media was examined with the influence exerted on the redox chemistry by cysteine evaluated in terms of the development of an analytical protocol. The electro-generation of o-quinone was found to be followed by a 1,4-addition reaction with available cysteine such that an increase in the current, attributed to the re-oxidation of the thiol-catechol adduct, could be exploited as means of quantifying the concentration of the thiol. The selectivity of the reaction has been assessed with no interference from lysine, tyrosine, methionine or cystine. Other amino acids possessing sulphydryl thiol functionalities (homocysteine and glutathione) were, however, found to react through a similar route to that observed with cysteine.     

Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine Cβ2

Background  

SCF ubiquitin ligases target numerous proteins for ubiquitin dependent proteolysis, including p27 and cyclin E. SCF and other cullin-RING ligases (CRLs) are regulated by the ubiquitin-like protein Nedd8 that covalently modifies the cullin subunit. The removal of Nedd8 is catalyzed by the Jab1/MPN domain metalloenzyme (JAMM) motif within the Csn5 subunit of the Cop9 Signalosome.     

The biosynthesis of the thiazole phosphate moiety of thiamin: the sulfur transfer mediated by the sulfur carrier protein ThiS

Thiamin-pyrophosphate is an essential cofactor in all living systems. The biosynthesis of both the thiazole and the pyrimidine moieties of this cofactor involves new biosynthetic chemistry. Thiazole-phosphate synthase (ThiG) catalyses the formation of the thiazole moiety of thiamin-pyrophosphate from 1-deoxy-D-xylulose-5-phosphate (DXP), dehydroglycine and the sulfur carrier protein (ThiS), modified on its carboxy terminus as a thiocarboxylate (ThiS-thiocarboxylate). Thiazole biosynthesis is initiated by the formation of a ThiG/DXP imine, which then tautomerizes to an amino-ketone. In this paper we study the sulfur transfer from ThiS-thiocarboxylate to this amino-ketone and trap a new thioenolate intermediate. Surprisingly, thiazole formation results in the replacement of the ThiS-thiocarboxylate sulfur with an oxygen from DXP and not from the buffer, as shown by electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) using (18)O labeling of the 13C-, 15N-depleted protein. These observations further clarify the mechanism of the complex thiazole biosynthesis in bacteria.     

Selective Electroanalytical Assay for Cysteine at a Boron Doped Diamond Electrode

The electrochemical oxidation of the cysteine‐quinone adduct has been examined as a means of providing an electroanalytical cysteine specific detection protocol. The appliance of square‐wave voltammetry allowed 0.5 μM as a limit of detection. The effects of various biologically relevant interferences including other thiols were studied and found to present no change in the voltammetric profile. The practical applicability and efficiency of the methodology was probed through the determination of cysteine concentration in growth tissue medium.     

Characterization by tandem mass spectrometry of stable cysteine sulfenic acid in a cysteine switch peptide of matrix metalloproteinases

Cysteine sulfenic acid (Cys-SOH) is an elusive intermediate in reactive oxygen species-induced oxidation reactions of many proteins such as peroxiredoxins and tyrosine phosphatases. Cys-SOH is proposed to play a vital role in catalytic and signaling functions. The formation of cysteine sulfinic acid (Cys-SO(2)H) and cysteine sulfonic acid (Cys-SO(3)H) has been implicated in the activation of matrix metalloproteinase-7 (MMP-7) and oxidation of thiol to cysteine sulfinic acid has been associated with the autolytic cleavage of MMP-7. We have examined the formation of cysteine sulfenic acid in a synthetic peptide PRCGVPDVA, which is a cysteine switch domain of MMP-7 and other matrix metalloproteases. We have prepared the cysteine sulfenic acid containing peptide, PRC(SOH)GVPDVA, by reaction with hydroxyl radicals generated by the Fenton reaction (Fe(+2)/H(2)O(2)). We characterized this modified peptide by tandem mass spectrometry and accurate mass measurement experiments. In addition, we used 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl) reagent to form an adduct with PRC(SOH)GVPDVA to provide additional evidence for the viability of PRC(SOH)GVPDVA in solution. We also characterized an intramolecular cysteine sulfinamide cross-link product PRC[S(O)N]GVPDVA based on tandem mass spectrometry and accurate mass measurement experiments. These results contribute to the understanding of a proteolytic cleavage mechanism that is traditionally associated with MMP activation.     

Thiamin biosynthesis in eukaryotes: characterization of the enzyme-bound product of thiazole synthase from Saccharomyces cerevisiae and its implications in thiazole biosynthesis

The biosynthesis of thiamin pyrophosphate in eukaryotes is different from the prokaryotic biosynthesis and is poorly understood to date. Only one thiazole biosynthetic gene has been identified (Thi4 in Saccharomyces cerevisiae). Here we report the identification and characterization of a Thi4-bound metabolite that consists of the ADP adduct of 5-(2-hydroxyethyl)-4-methylthiazole-2-carboxylic acid. The unexpected structure of this compound yields the first insights into the mechanism of thiamin thiazole biosynthesis in eukaryotes.     

Characterization of the nucleophilic reactivities of thiocarboxylate, dithiocarbonate and dithiocarbamate anions

The kinetics of the reactions of thiocarboxylate and thiocarbonate anions with benzhydrylium ions have been determined in acetonitrile solution using laser-flash photolytic techniques. The second-order rate constants (k) correlate linearly with the electrophilicity parameters E of the benzhydrylium ions, as required by the correlation log k (20 °C) = s(N)(N + E) (J. Am. Chem. Soc., 2001, 123, 9500-9512), allowing us to calculate the nucleophile-specific parameters N and s(N) for these anions. With these parameters, a direct comparison of the reactivities of thiocarboxylate, dithiocarbonate and dithiocarbamate anions with other nucleophiles becomes possible.     

Isopenicillin N synthase mediates thiolate oxidation to sulfenate in a depsipeptide substrate analogue: implications for oxygen binding and a link to nitrile hydratase?

Isopenicillin N synthase (IPNS) is a nonheme iron oxidase that catalyzes the central step in the biosynthesis of beta-lactam antibiotics: oxidative cyclization of the linear tripeptide delta-L-alpha-aminoadipoyl-L-cysteinyl-D-valine (ACV) to isopenicillin N (IPN). The ACV analogue delta-L-alpha-aminoadipoyl-L-cysteine (1-(S)-carboxy-2-thiomethyl)ethyl ester (ACOmC) has been synthesized as a mechanistic probe of IPNS catalysis and crystallized with the enzyme. The crystal structure of the anaerobic IPNS/Fe(II)/ACOmC complex was determined to 1.80 A resolution, revealing a highly congested active site region. By exposing these anaerobically grown crystals to high-pressure oxygen gas, an unexpected sulfenate product has been observed, complexed to iron within the IPNS active site. A mechanism is proposed for formation of the sulfenate-iron complex, and it appears that ACOmC follows a different reaction pathway at the earliest stages of its reaction with IPNS. Thus it seems that oxygen (the cosubstrate) binds in a different site to that observed in previous studies with IPNS, displacing a water ligand from iron in the process. The iron-mediated conversion of metal-bound thiolate to sulfenate has not previously been observed in crystallographic studies with IPNS. This mode of reactivity is of particular interest when considered in the context of another family of nonheme iron enzymes, the nitrile hydratases, in which post-translational oxidation of two cysteine thiolates to sulfenic and sulfinic acids is essential for enzyme activity.