Isotope Incorporation Experiments Research Articles

Quinone-mediated extracellular electron transfer processes in ex situ biomethanation reactors

Redox mediators are used in a wide diversity of systems including biological ones. We investigated the effect of adding an artificial quinone (2,7-AQDS) as external redox molecule to an anaerobic digester system dominated by hydrogenotrophic methanogens. When oxidized AQDS was present, the methanogens diverted electrons from H2 to reduce AQDS instead of CO2. The AQDS reduction process was accompanied by a temporary CH4 inhibition, which was re-established several days after the full reduction of AQDS to AH2QDS. The presence of AQDS furthermore resulted in a community shift from Methanobacterium as the dominant methanogen to a more diverse community of methanogens. Protein expression profiles showed a shift in cofactor preference of the adapted community, as a potential response mechanism to AQDS inhibition. AH2QDS was only used as electron donor to a limited extent. Stable isotope incorporation experiments here indicated that the acetogen Acetoanaerobium used AH2QDS to reduce CO2 into acetate.

Dendrillic Acids A and B: Nitrogenous, Rearranged Spongian Nor-Diterpenes from a Dendrilla sp. Marine Sponge.

Two nitrogenous rearranged spongian nor-diterpenoids, dendrillic acids A and B, were isolated from a marine sponge Dendrilla sp. (order: Dendroceratida; family: Darwinellidae). The structures of the metabolites were elucidated on the basis of spectroscopic analysis as well as density functional theory prediction of NMR chemical shifts and application of the DP4+ algorithm. The absolute configuration of the metabolites was established via comparison of experimental and time-dependent density functional theory predicted electronic circular dichroism data. An unusual epimerization reaction was observed leading to the interconversion of the metabolites upon storage in dimethyl sulfoxide solution, which is proposed to proceed via an anionic pathway as probed via isotopic incorporation experiments. Evaluation against a panel of micro-organisms and cell lines revealed that the compounds were devoid of any significant biological activity against all organisms tested, with the exception of mild antiprotozoal activity displayed by dendrillic acid B (2) against Giardia duodenalis.

Identification of 7-Deoxy-desulfo-argino-cylindrospermopsin, the Missing Piece in Cylindrospermopsin Biosynthesis.

Cylindrospermopsin, a major cyanotoxin, is produced by freshwater cyanobacteria. Its biosynthesis starts from arginine and glycine and involves five polyketide synthases and several tailoring enzymes. We report the identification of 7-deoxy-desulfo-argino-cylindrospermopsin in several cylindrospermopsin-producing cyanobacteria using mass spectrometry experiments. We have purified this new metabolite and established its structure by 1D and 2D NMR spectroscopy using scalar-based 1H-1H, 1H-13C, and 1H-15N as well as 2D 1H-1H ROESY correlation experiments. Using labeled arginines in isotopic incorporation experiments, we have shown that arginine is fully incorporated into 7-deoxy-desulfo-argino-cylindrospermopsin and that the uracil ring of cylindrospermopsin originates from the guanidino moiety of arginine, thus solving a long-standing puzzling question. CyrG and CyrH from the cylindrospermopsin-producing Oscillatoria sp. PCC 6506 were overproduced in Escherichia coli and purified to homogeneity. We showed that CyrG is a zinc-dependent hydrolase, homologous to adenosine deaminases, that transforms 7-deoxy-desulfo-argino-cylindrospermopsin into 7-deoxy-desulfo-cylindrospermopsin and ornithine, with the following kinetic parameters: KM = 0.21 ± 0.05 μM and kcat = 0.19 ± 0.02 min-1. CyrG contained 0.55 mol of zinc per mol of monomer but could be activated by FeII or CoII. CyrH contained almost no metal and showed no such activity even in the presence of excess metal. Using structure-based alignments and secondary structure predictions, we propose that the fifth and last polyketide synthase CyrF in cylindrospermopsin biosynthesis contains an unprecedented C-terminal domain homologous to N-acetyltransferases. We suggest that this domain catalyzes the condensation of the CyrF product with arginine to give 7-deoxy-desulfo-argino-cylindrospermopsin. This would be an unprecedented termination step for a polyketide synthase.

Characterization of Highly Reductive Modification of Tetracycline D-Ring Reveals Enzymatic Conversion of Enone to Alkane

Tetracyclines are an eminent family of type II polyketides that possess a variety of decoration on the skeletons. However, apart from the oxidative modification in aureolic acid compounds, there are few cases of the further conversion of α,β-unsaturated ketones in the tetracycline D-ring. Here, we identified two reductases (TjhO5 and TjhD4), which can highly reduce the α,β-unsaturated ketone of the D-ring in unconventional tetracyclines. By identifying related intermediates and conducting isotope incorporation experiments, we demonstrated that the entire transformation could be accomplished by TjhO5 and TjhD4 collectively via two distinct pathways involving different enzymatic mechanisms. A distinctive deoxygenation mechanism was possibly involved in the TjhO5-mediated continuous reduction of C═O to CH2. These findings highlight the unusual post-modification of tetracyclines and facilitate further engineering and biocatalysis to enrich the structural diversities.

Biosynthesis of Chlorinated Lactylates in Sphaerospermopsis sp. LEGE 00249.

Lactylates are an important group of molecules in the food and cosmetic industries. A series of natural halogenated 1-lactylates, chlorosphaerolactylates (1–4), were recently reported from Sphaerospermopsis sp. LEGE 00249. Here, we identify the cly biosynthetic gene cluster, containing all the necessary functionalities for the biosynthesis of the natural lactylates, based on in silico analyses. Using a combination of stable isotope incorporation experiments and bioinformatic analysis, we propose that dodecanoic acid and pyruvate are the key building blocks in the biosynthesis of 1–4. We additionally report minor analogues of these molecules with varying alkyl chains. This work paves the way to accessing industrially relevant lactylates through pathway engineering.

Biosynthesis of Anatoxins in Cyanobacteria: Identification of the Carboxy-anatoxins as the Penultimate Biosynthetic Intermediates.

Anatoxin-a, homoanatoxin-a, and dihydroanatoxin-a are potent cyanobacterial neurotoxins. They are biosynthesized in cyanobacteria from proline and acetate by a pathway involving three polyketide synthases. We report the identification of carboxy-anatoxin-a, carboxy-homoanatoxin-a, and carboxy-dihydroanatoxin-a in acidic extracts of Cuspidothrix issatschenkoi CHARLIE-1, Oscillatoria sp. PCC 6506, and Cylindrospermum stagnale PCC 7417, respectively, using liquid chromatography coupled to mass spectrometry. The structure of these carboxy derivatives was confirmed by mass spectrometry and by isotopic incorporation experiments using labeled proline and acetate. Each of these three cyanobacteria only produce one carboxy-anatoxin, suggesting that these metabolites are the product of the hydrolysis by AnaA, the type II thioesterase, of the thioesters bound to AnaG, the last polyketide synthase of the pathway. By measuring the rate of isotopic incorporation of labeled proline into carboxy-homoanatoxin-a and homoanatoxin-a produced by Oscillatoria sp. PCC 6506, we show that carboxy-homoanatoxin-a is the intracellular precursor of homoanatoxin-a, and that homoanatoxin-a is then excreted into the extracellular medium. The transformation of carboxy-homoanatoxin-a into homoanatoxin-a is a very slow two-step process, with accumulation of carboxy-homoanatoxin-a, suggesting that the decarboxylation is spontaneous and not enzymatically catalyzed. However, an unidentified and extracellular catalyst accelerates the decarboxylation when the cell extracts are prepared at neutral pH.

Hydroxyl regioisomerization of anthracycline catalyzed by a four-enzyme cascade

Ranking among the most effective anticancer drugs, anthracyclines represent an important family of aromatic polyketides generated by type II polyketide synthases (PKSs). After formation of polyketide cores, the post-PKS tailoring modifications endow the scaffold with various structural diversities and biological activities. Here we demonstrate an unprecedented four-enzyme-participated hydroxyl regioisomerization process involved in the biosynthesis of kosinostatin. First, KstA15 and KstA16 function together to catalyze a cryptic hydroxylation of the 4-hydroxyl-anthraquinone core, yielding a 1,4-dihydroxyl product, which undergoes a chemically challenging asymmetric reduction-dearomatization subsequently acted by KstA11; then, KstA10 catalyzes a region-specific reduction concomitant with dehydration to afford the 1-hydroxyl anthraquinone. Remarkably, the shunt product identifications of both hydroxylation and reduction-dehydration reactions, the crystal structure of KstA11 with bound substrate and cofactor, and isotope incorporation experiments reveal mechanistic insights into the redox dearomatization and rearomatization steps. These findings provide a distinguished tailoring paradigm for type II PKS engineering.

Proteome Scale-Protein Turnover Analysis Using High Resolution Mass Spectrometric Data from Stable-Isotope Labeled Plants.

Protein turnover is an important aspect of the regulation of cellular processes for organisms when responding to developmental or environmental cues. The measurement of protein turnover in plants, in contrast to that of rapidly growing unicellular organismal cultures, is made more complicated by the high degree of amino acid recycling, resulting in significant transient isotope incorporation distributions that must be dealt with computationally for high throughput analysis to be practical. An algorithm in R, ProteinTurnover, was developed to calculate protein turnover with transient stable isotope incorporation distributions in a high throughput automated manner using high resolution MS and MS/MS proteomic analysis of stable isotopically labeled plant material. ProteinTurnover extracts isotopic distribution information from raw MS data for peptides identified by MS/MS from data sets of either isotopic label dilution or incorporation experiments. Variable isotopic incorporation distributions were modeled using binomial and beta-binomial distributions to deconvolute the natural abundance, newly synthesized/partial-labeled, and fully labeled peptide distributions. Maximum likelihood estimation was performed to calculate the distribution abundance proportion of old and newly synthesized peptides. The half-life or turnover rate of each peptide was calculated from changes in the distribution abundance proportions using nonlinear regression. We applied ProteinTurnover to obtain half-lives of proteins from enriched soluble and membrane fractions from Arabidopsis roots.

The Phormidolide Biosynthetic Gene Cluster: A trans-AT PKS Pathway Encoding a Toxic Macrocyclic Polyketide.

Phormidolide is a polyketide produced by a cultured filamentous marine cyanobacterium and incorporates a 16-membered macrolactone. Its complex structure is recognizably derived from a polyketide synthase pathway, but possesses unique and intriguing structural features that prompted interest in investigating its biosynthetic origin. Stable isotope incorporation experiments confirmed the polyketide nature of this compound. We further characterized the phormidolide gene cluster (phm) through genome sequencing followed by bioinformatic analysis. Two discrete trans-type acyltransferase (trans-AT) ORFs along with KS-AT adaptor regions (ATd) within the polyketide synthase (PKS) megasynthases, suggest that the phormidolide gene cluster is a trans-AT PKS. Insights gained from analysis of the mode of acetate incorporation and ensuing keto reduction prompted our reevaluation of the stereochemistry of phormidolide hydroxy groups located along the linear polyketide chain.

Biosynthesis of Athmu, a α,γ-hydroxy-β-amino acid of pahayokolides A–B

Pahayokolides A–B are cyanobacteria derived non-ribosomal peptides which exhibit cytotoxicity against a number of cancer cell lines. The biosynthetic origin of the 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid (Athmu) moiety has been investigated using stable isotope incorporation experiments. While α-ketoisocaproic acid (α-KIC), α-hydroxyisocaproic acid (α-HIC), and leucine all serve as precursors to Athmu, the feeding of [1-13C] α-KIC results in more than threefold greater 13C enrichment than the other precursors. This result suggests that α-KIC is the immediate precursor which is selected and activated by the adenylation domain of the loading NRPS module and subsequently reduced in a fashion similar to that of the recently identified pathways for cryptophycins A–B, cereulide, and valinomycin.

ChemInform Abstract: A New Combined Source of “CN” from N,N‐Dimethylformamide and Ammonia in the Palladium‐Catalyzed Cyanation of Aryl C—H Bonds.

AbstractIsotopic incorporation experiments reveal that the carbon and nitrogen of the “CN” originate from the N,N‐dimethyl moiety of DMF and ammonia, respectively.

Growth and catabolism in isotopic incorporation: a new formulation and experimental data

Summary 1. We propose a new model that uses an organism’s change in mass to estimate isotope incorporation rates. Our model is a re‐parameterization of a widely used one‐compartment‐first‐order kinetic model that estimates the rate of isotopic incorporation as a function of time. If animals are growing exponentially and fractional growth rates are measured, the time model allows partitioning the contribution of growth and catabolic turnover to isotopic incorporation. The model can be extended to include more than one‐compartment. 2. Our model makes the same assumptions but uses mass change as an independent variable. It estimates the ratio of the contribution of catabolic turnover to growth. 3. To test the model, we fed juvenile Nile tilapia (Oreochromis niloticus) at three different rations and measured their change in mass and tissue δ13C isotopic composition for 128 days. 4. Fish grew exponentially and fractional growth rates differed significantly between rations. The contribution of catabolism (tissue replacement) to isotopic incorporation was 2·6 to 5 times higher than growth (tissue addition) in liver. In muscle tissue, the contribution of catabolism and growth were roughly equivalent for all rations. 5. When we used the parameters of the mass model to re‐parameterize the time model we found a relatively good fit (r2 = 0·8 ± 0·1, mean ± SD) to the isotopic incorporation data. 6. The mass model estimated a smaller, and more biologically realistic range of δ∞ values than did the time model which suggests that it might yield better results when isotopic incorporation experiments are not carried on long enough to yield estimates of the asymptotic isotopic value of an animal’s tissues after a diet shift. 7. Rather than being alternatives, the time and mass models are complementary in that they allow us to see the same phenomenon from different perspectives.

A New Combined Source of “CN” from N,N-Dimethylformamide and Ammonia in the Palladium-Catalyzed Cyanation of Aryl C−H Bonds

An unprecedented protocol for cyanation at arene C-H bonds has been developed by employing N,N-dimethylformamide and ammonia as a combined source for the cyano "CN" unit. Isotopic incorporation experiments revealed that the carbon and nitrogen of the "CN" originate from the N,N-dimethyl moiety of DMF and ammonia, respectively. The present cyanation reaction shows an excellent degree of regioselectivity, producing only monosubstituted nitriles at the less hindered C-H position, and it allows for the preparation of doubly labeled nitrile compounds for the first time.

Tyl1a, a TDP‐6‐deoxy‐D‐xylo‐4‐hexulose 3,4‐isomerase from Streptomyces fradiae: Structure Prediction, Mutagenesis and Solvent Isotope Incorporation Experiments to Investigate Reaction Mechanism

Understanding the structure and mechanism of sugar nucleotide processing enzymes is invaluable in the generation of designer enzymes for biotransformation, for instance, in connection with engineering antibiotic glycosylation. In this study, homology modelling and mechanistic comparison to the structurally related RmlC epimerase family has been used to identify and assign functions to active-site residues in the Tyl1a-catalysed keto-sugar nucleotide isomerisation process. Tyl1a His63 is implicated as the base that initiates the isomerisation process by substrate C-3 deprotonation, with Arg109 stabilising the resulting enolate. Subsequent O-3 deprotonation (potentially by His65) and C-4 protonation (potentially by Tyr49) complete the isomerisation process.

Cover Picture: Tyl1a, a TDP‐6‐deoxy‐D‐xylo‐4‐hexulose 3,4‐isomerase from Streptomyces fradiae: Structure Prediction, Mutagenesis and Solvent Isotope Incorporation Experiments to Investigate Reaction Mechanism (ChemBioChem 8/2008)

The cover picture shows the modelled structure of Streptomyces fradiae Tyl1a and the isomerisation reaction it catalyses en route to the biosynthesis of the antibiotic tylosin. Specifically, Tyl1a is a TDP-6-deoxy-D-xylo-4-hexulose 3,4-isomerase. Using a combination of homology modelling, site-directed mutagenesis and solvent isotope incorporation experiments, we have identified potential catalytic residues of Tyl1a. This enzyme is predicted to be dimeric, with the core of each subunit comprised of a cupin-like β-barrel where catalysis takes place. A single β-barrel is shown, with the catalytic residues depicted in full atom representation. This study has enabled us to postulate a reaction mechanism for Tyl1a that is similar to that of the structurally related RmlC family of ketosugar nucleotide epimerases. For more details, see the article by R. Field, D. M. Lawson et al. on p. 1295 ff.

Beyond the reaction progress variable: the meaning and significance of isotopic incorporation data

Ecologists conduct isotopic incorporation experiments to determine the residence time of various stable isotopes in animal tissues. These experiments permit determining the time window through which isotopic ecologists perceive the course of diet changes, and therefore the scale of the inferences that we can make from isotopic data. Until recently, the results of these experiments were analyzed using first-order, one-compartment models. Cerling et al. (Oecologia 151:175-189, 2007) proposed an approach they named the reaction progress variable to: (1) determine how many compartments are needed to describe a pattern of istopic incorporation, and (2) to estimate the size and rate constant of each pool. We elaborate on the approach described by Cerling et al. (Oecologia 151:175-189, 2007) by providing a way to estimate average retention times for an isotope in a tissue (and its associate error) for multi-compartment models. We also qualify the interpretation of the parameters in multi-compartment models by showing that many possible mechanisms yield models with the same functional form. Multi-compartment models are phenomenological, rather than mechanistic descriptions, of incorporation data. Finally, we propose the use of information theoretic criteria to assess the number of compartments that must be included in models of isotopic incorporation.

Biosynthetic studies of the DSP toxin skeleton.

Marine toxins have drawn wide interest because their economical impact and disastrous effect upon the shellfish industry and public health in many parts of the world. One of the most interesting group of substances of marine toxins, from structural and pharmacological points of view are polyether compounds, which generally present a great diversity in size and potent biological activities. The subject of this work was about to biosynthesis of okadaic acid skeleton as leader as DSP toxins. Its biosynthesis attracts considerable attention since the carbon skeleton has been shown to be synthesised via an unusual route. In this paper we report on stable isotope incorporation experiments on DSP toxin in artificial cultures of dinoflagellate. The comparison of the degrees of incorporation in these samples measured by different methods led to contradictory results. This implies that further experimental data is needed in order to propose a logical biogenetic scheme.

Antibody catalysis of the oxidation of water.

Recently we reported that antibodies can generate hydrogen peroxide (H2O2) from singlet molecular oxygen (1O2*). We now show that this process is catalytic, and we identify the electron source for a quasi-unlimited generation of H2O2. Antibodies produce up to 500 mole equivalents of H2O2 from 1O2*, without a reduction in rate, and we have excluded metals or Cl- as the electron source. On the basis of isotope incorporation experiments and kinetic data, we propose that antibodies use H2O as an electron source, facilitating its addition to 1O2* to form H2O3 as the first intermediate in a reaction cascade that eventually leads to H2O2. X-ray crystallographic studies with xenon point to putative conserved oxygen binding sites within the antibody fold where this chemistry could be initiated. Our findings suggest a protective function of immunoglobulins against 1O2* and raise the question of whether the need to detoxify 1O2* has played a decisive role in the evolution of the immunoglobulin fold.

Biosynthesis of bitter acids in hops. A (13)C-NMR and (2)H-NMR study on the building blocks of humulone.

The biosynthesis of humulone, an antibacterial bitter acid from hops, was studied by isotope-incorporation experiments using (13)C-labelled glucose or (2)H(2)O. (13)C enrichments, (2)H enrichments and (13)C(13)C coupling patterns identify isovaleryl-CoA, malonyl-CoA and dimethylallyl pyrophosphate as precursors for humulone. Dimethylallyl pyrophosphate, which serves as a building block for the bitter acid, is generated via the deoxyxylulose pathway of terpenoid biosynthesis. The data confirm that a symmetrical intermediate is involved in humulone formation.

Evidence for de novo biosynthesis of the polyketide fragment of diaulusterol A by the northeastern pacific dorid nudibranch diaulula sandiegensis

Stable isotope incorporation experiments using sodium [1, 2-13C2]acetate have demonstrated that the 3-hydroxybutyrate substituent of diaulusterol A (1) is biosynthesized de novo by the dorid nudibranch Diaulula sandiegensis. There was no evidence for acetate incorporation into the steroid portion of 1, nor were radiolabeled mevalonate or cholesterol incorporated.