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In situ proton NMR analysis of a-alkynoate biotransformations From ‘invisible’ substrates to detectable metabolites Lothar Brecker 1, *, Julia Petschnigg 1 , Nicole Depine ´ 1 , Hansjo¨ rg Weber 1 and Douglas W. Ribbons 1,2,† 1 Institute of Organic Chemistry, University of Technology Graz, Austria; 2 Institute of Biotechnology, University of Technology Graz, Austria Only 2% of the known natural products with acetylenic bonds are a-alkynoates. Their polarized, conjugated triple bond is an optimal target for an enzymic hydration. Therefore they are good substrates for the enzymes involved in metabolism of acetylenic compounds, resulting in products that are suitable for bacterial growth. We isolated a Pseudomonas putida strain growing on 2-butynedioate as well as on propynoate, and determined the metabolic pathways of these two a-alkynoates. The triple bonds in both compounds were initially hydrated and 2-ketobutandioate as well as 3-ketopropanoate were formed. These two b-keto acids were decarboxylated resulting in pyruvate and acetaldehyde, respectively. Pyruvate was further hydrolysed mainly to acetate and formate, whereas minor amounts were reduced to lactate. In the other biotransformation, acetaldehyde was oxidized to acetate accompanied by the reduction of 3-ketopro- panoate to 3-hydroxypropanoate. Analyses of these meta- bolic processes were performed by in situ 1 H-NMR spectroscopy in 1 H 2 O, although the substrates, propynoate and 2-butynedioate, carried only one or even no detectable protons, respectively. However, while protons from the solvent are incorporated in the course of the pathway, the metabolites can be detected and identified. Therefore a detailed determination of the metabolic process is possible. Keywords: 2-butynedioate; in situ 1 H-NMR; Pseudomonas putida; propynoate; triple bond. Acetylenic bonds are quite rare in natural compounds compared with vinyl bonds, carbonyl groups, carboxylates or aromatic rings, but they are found in more natural products than bromides, chlorides, or nitriles [1]. Com- pounds containing triple bonds are widely distributed in plants, fungi, bacteria, and other living organisms [1]. In addition to these natural compounds, several synthetic chemicals contain triple bonds. The amount of these products released to the natural environment in the form of drugs, pesticides, or even accidentally can only be speculated upon. While several natural and unnatural acetylenic compounds possess high toxic potential [1], it is of interest to elucidate their pathways in bacterial metabo- lism and detoxification. However, these biodegradations have not yet been generally studied and therefore only a small number of enzymes metabolizing triple bonds have been described. Acetylene, the simplest compound with a triple bond, is reported to be reduced to ethylene by nitrogenases (E.C. 1.18.6.1 [2]), or to be hydrated by acetylene hydratase (E.C. 4.2.1.71 [3]). Triple bonds in other substrates, however, are isomerized to conjugated allenes (E.C. 5.3.3.8 [4–6]), or hydrated (E.C. 4.2.1.71 [7–10]). In searching for organisms that degrade a-alkynoates, we isolated a Pseudomonas putida strain growing on 2-butyne- dioate or propynoate as sole carbon source. To investigate the acetylene bond biodegradation, we used in situ proton nuclear magnetic resonance ( 1 H-NMR) in water ( 1 H 2 O) as a versatile analytical method [11–15]. This technique allows 1 H-NMR spectra to be directly recorded at any stage of a biotransformation with or without sampling the culture, and enables the identification of metabolites, examination of metabolic pathways, and analysis of the fermentation time courses [11–15]. While, except in propynoate, the triple bond in a-alkynoates do not carry a proton, the substrates are ÔinvisibleÕ in 1 H-NMR. The incorporation of protons from the solvent in the pathway, however, makes the metabolites detectable. Therefore additional defined amounts of deuterium (D 2 O) in water ( 1 H 2 O) can easily be detected in the metabolites and allow conclusions about detailsofthepathway[16].Herewedescribethein situ 1 H-NMR analysis of the 2-butynedioate and propynoate biotransformations by the isolated P. putida strain. Both pathways were found to be initiated by a hydrolysis of the triple bond. Correspondence to L. Brecker, Institute of Organic Chemistry, University of Technology Graz, Stremayrgasse 16, A-8010 Graz, Austria. Fax: + 43 316 873 8740, Tel.: + 43 316 873 8250, E-mail: joerg@orgc.tu-graz.ac.at or lothar.brecker@univie.ac.at Enzymes: acetylene hydratase (acetylenecarboxylate hydratase) [E.C. 4.2.1.71]; acetylene isomerase (dodecenoyl-CoA delta-isomerase) [E.C. 5.3.3.8]; nitrogenase [E.C. 1.18.6.1]. *Present address: Institute of Organic Chemistry, University Vienna, Wa ¨ hringer Straße 38, A-1090 Wien, Austria. Note: Deceased October 7, 2002. This paper is dedicated to his memory. His enthusiasm, insights, and unique perspective were an inspiration to many, and his presence is greatly missed. (Received 21 October 2002, revised 5 January 2003, accepted 14 January 2003) Eur. J. Biochem. 270, 1393–1398 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03460.x Materials and methods Chemicals All chemicals were purchased from Sigma-Aldrich Chemi- cal Co. in the highest available purity and used without further purification. Strain and media A strain was isolated from rotten fruits by growth on mineral medium at pH 7 and 30 °C [17] using 2-butyne- dioate (3 m M ) as sole carbon source. It was identified as P. putida (DSMZ ID 99–842) by ÔDeutsche Stammsam- mlung von Mikroorganismen, Braunschweig, GermanyÕ. Growth on other substrates was tested on mineral agar plates [17] containing the substrates (5 m M )atpH7. Liquid cultures were grown on a rotary shaker at 30 °C and pH 7 in mineral medium [17] supplemented with 5m M 2-butynedioate or 5 m M propynoate, respectively. Samples were taken every hour for determination of the optical density (D 570 ). After 24 h cells were centrifuged, washed, and resuspended in 100 m M phosphate buffer in 1 H 2 O (pH 7) or 1 H 2 O/D 2 O (1 : 1, pH 7) to a suspen- sion with a D 570 of 0.3–0.5. Aerobic biotransformations to determine the metabolism time courses at 30 °C were started by addition of the substrate (5 m M )to these suspensions. They were sampled at every 2 h for UV measurements and then every 12 h for 1 H-NMR analysis. UV spectroscopy UV spectra were taken on a Spectronic Genesis 2PC, Thermo Spectronic, Rochester, USA and with a Shimadzu 240, Shimadzu, Kyoto, Japan. Bacterial growth rates were determined by measuring the optical density of cell cultures at 570 nm (D 570 ). Substrate consumption was determined from the supernatant of the culture after centrifugation of the cells. Spectra were measured from 220 nm to 320 nm. 1 H-NMR spectroscopy All 1 H-NMR spectra were recorded on a 200-MHz narrow bore magnet (Gemini 2000, Varian, Palo Alto, CA, USA) equipped with a 5-mm broadband probe head. For a lock a D 2 O vortex capillary was added to the NMR tube to avoid 1 H/D exchange reactions. During measurements the tube was rotated at 20 rev.Æs )1 .The huge water signal was suppressed using the presaturation method [18,19]. The following measurement parameters were adjusted: presaturation duration, 1.0 s; 1 H pulse angle, 90°; acquisition time, 2.0 s; relaxation delay, 1.5 s. A total of 128 scans was accumulated and after a zero filling to 32 768 data points the free induction decay was Fourier transformed. The 3-(trimethylsilyl)-propionic- 2,2,3,3-d 4 acid (TSP) signal ( 1 H d: 0.00 p.p.m) was used as an external reference. For in situ 1 H-NMR measure- ment we analysed the supernatant of the cell cultures. All metabolites were identified by addition of standard solutions to the sample. Results Isolation and characterization of the strain We isolated a strain from rotten fruits by aerobic enrichment cultures in liquid minimal medium containing 2-butynedio- ate as the sole carbon source. The bacterial strain was classified as P. putida (DSMZ ID 99–842) by ÔDeutsche Stammsammlung von Mikroorganismen, Braunschweig, GermanyÕ.TheP. putida strain accepted several other acetylenic and nonacetylenic compounds as substrates for growth. Apart from the triple bonds and carboxylate functions these compounds additionally contained hydroxyl groups, keto groups, or an aromatic ring (Table 1). None of these chemical functionalities significantly inhibited bacterial growth, which was monitored by following D 570 (cell density) (Fig. 1). Growth rates are comparable for both investigated aerobic biotransformations, indicating that the presence of one or two carboxyl groups does not considerably influence the substrate acceptance. To determine the metabolic Table 1. Growth of P. putida on different substrates, measured on agar plates. Substrate Structure Growth a Propynoate + 2-Butynedioate + Propynol ++ But-3-yn-1-ol + Pent-3-yn-1-ol + Phenylethyne b + Pyruvate + Succinate +++ Citrate +++ Glucose +++ a +, Comparable to growth rate on 2-butyndioate; ++, 2–3 times faster growth rate than on 2-butyndioate; +++, 5–10 times faster growth rate than on 2-butyndioate. b Phenylethynes are substrates for ring dioxygenases in other bacteria [7]. 1394 L. Brecker et al. (Eur. J. Biochem. 270) Ó FEBS 2003 pathways of the two acetylenic compounds UV spectroscopy and in situ 1 H-NMR [11–15] were used. Biotransformation of propynoate The metabolism of propynoate (k max ¼ 263 nm) was first analysed by UV indicating that the substrate concentration (5 m M ) in the supernatant decreased during the first 12 h and that no significant accumulation of a UV absorbing metabolite occurred (Fig. 2). The substrate consumption was confirmed by in situ 1 H-NMR analysis ( 1 H d: 3.06 p.p.m) in 1 H 2 O. Spectra indicated the additional presence of a small amount of 3-ketopropanoate ( 1 H d: 3.48 p.p.m) and acetaldehyde ( 1 H d: 1.17 p.p.m) during the propynoate consumption. Both compounds were only identified in the keto form, as the corresponding hydrates were present in concentrations below the limit of detection. The detected transient metabolites were present at  0.2– 0.4 m M (Fig. 3a and b). In parallel the amount of acetate formed ( 1 H d: 1.90 p.p.m) and 3-hydroxypropanoate ( 1 H d: 2.42 p.p.m) increased up to 2.5 m M while propynoate was metabolized completely. Whereas acetate was further con- sumed and metabolized to non-detectable products during the following 64 h, 3-hydroxypropanoate had not been accepted as a substrate and was present at a constant concentration (Fig. 3c). These findings indicate an initial hydration of the triple bond in propynoate forming 3-hydroxyprop-2-enoate acid. This metabolite spontaneously isomerizes to 3-ketopropa- noic acid, which is than partly decarboxylated forming acetaldehyde and gaseous carbon dioxide. In the following step, acetaldehyde was oxidized to acetate accompanied by the reduction of 3-ketopropanoate to 3-hydroxypropanoate. These parallel reactions suggest that hydrogen atoms from acetaldehyde are incorporated into the alcohol formation in the other metabolite. The assumption was proved by adding acetaldehyde (0.5 m M and 5 m M ) to the biotransformation. The 0.5 m M amount led to a direct consumption of the acetaldehyde added and the production of equal amounts of 3-hydroxypropanoate, while the concentration of transient 3-ketopropanoate was too low to be detected. Addition of 5m M acetaldehyde obviously induced other enzymes that metabolise this substrate in a different way. Performing the propynoate biotransformation in 1 H 2 O/ D 2 O (1 : 1) led to an incorporation of 50% deuterium in all metabolites. The addition of 0.5 m M acetaldehyde to this biotransformation in 50% D 2 Ocausedan 10% higher amount of hydrogen in the acetate, as it is formed directly from the acetaldehyde. The incorporation of a higher hydrogen amount in position three of 3-hydroxypropanoate was not determined, probably due to isotopic exchange during the reduction/oxidation reactions. The propynoate pathway in P. putida isshowninFig.4. Biotransformation of 2-butynedioate The UV spectrophotometric analysis of the 2-butynedioate metabolism provided scattered absorptions at k max ¼ 265 nm during the first 24 h of the biotransformation. Furthermore the consumption of 2-butynedioate could not be monitored by 1 H-NMR due to the lack of protons in this substrate. However, 1 H-NMR clearly indicates the formation of pyruvic acid [ 1 H d: 2.37 p.p.m. (keto form); Fig. 1. Aerobic growth of isolated P. putida on 2-butynedioate (j) and propynoate (d). Fig. 2. UV Spectra taken from the supernatant of the propynoate fer- mentation. Spectra were taken every 2 h and indicate metabolism of the conjugated system in propynoate. Ó FEBS 2003 1 H-NMR of a-alkynoate biotransformations (Eur. J. Biochem. 270) 1395 1.43 p.p.m. (hydrate form)] during this time period. The accumulation of this metabolite explains the UV results, because it absorbs in the same spectral range as 2-butyne- dioate. While no intermediate with four carbon atoms was detected, we assumed that pyruvate was formed by decarb- oxylation of 2-ketobutandioate, the product of a triple-bond hydrolysis of 2-butynedioate. An initial decarboxylation was excluded, as neither propynoate, nor its metabolite 3-hydroxypropanoate were detected. Control experiments using 2-ketobutandioate as substrate confirmed this assumption. The decarboxylation by the P. putida strain was about 10 times faster than the spontaneous decarboxy- lation, indicating the presence of an induced decarboxylase in the organism. The accumulated pyruvate was mainly hydrolysed to equal amounts of acetate ( 1 H d: 1.87 p.p.m) and formate ( 1 H d: 8.36 p.p.m), which were both further slowly metabolized to nondetectable products (Fig. 5). The small shift differences of the acetate signal in the two biotransformations were due to variations in the salt concentrations and the pH value [16]. About 10% of the pyruvate was transformed to lactate ( 1 H d: 1.29 p.p.m.; Fig. 5), indicating an incorporation of hydrogen from formate degradation. A metabolism of pyruvate via a dehydrogenase might also be possible in small amounts. Fig. 6 shows the 2-butynedioate metabolic pathway in P. putida. Discussion Of the variety of isolated natural products with acetylenic bonds only 2% are a-alkynoates [1]. As these compounds are seldomly accumulated, they seem to be good substrates for metabolism of the triple bond. So far only one hydratase from a Pseudomonas strain has been described to act directly on a-alkynoates [9,10]. It is reported to accept 2-butyne- dioate and propynoate as substrates. One b-alkynoate (3-butynoate) has also been described to be hydrated by another hydratase from Pseudomonas BB1 [8]. However, none of these hydratases has been purified. Our isolation again resulted in a Pseudomonas strain that grew on 2-butynedioate and propynoate. Although using strictly aerobic conditions in both cases, the triple bonds were hydrolysed, and not oxidized. The two triple bonds in the substrates were probably hydolysed by the same enzyme, Fig. 4. Propynoate metabolic pathway in the isolated P. putida strain. An initial hydrolysis formed 3-ketopropanoate, which was then partly decarboxylated to acetaldehyde. The latter was dehydrogenated to acetate, whereas the 3-ketopropanoate was hydrated to 3-hydroxy- propanoate. Fig. 3. Selected 1 H-NMR spectra from propynoate metabolism. (A) Spectrum of the substrate. (B) Spectrum after 36 h. Small amounts of acetealdehyde and 3-ketopropanoate were identified by the addition of standard solutions. Larger amounts of acetate and 3-hydroxypro- panoate were detected directly from the spectra. Unidentified minor by-products are indicated with asterisks (*). (C) Spectrum after 128 h. Starting material and intermediates were completely consumed and 3-hydroxypropanoate accumulated; some acetate was left and slowly further metabolized. 1396 L. Brecker et al. (Eur. J. Biochem. 270) Ó FEBS 2003 resulting in different metabolites. Whereas the metabolites of the dicarboxylate were completely consumed, half of the substrate with the terminal acetylenic bond was transformed to a compound that was not further accepted as a substrate. This finding seems to be in contrast with the bacterial growth on the two substrates, which resulted in a similar optical density. However, the discrepancy is explainable considering that a-alkynoates are probably not the natural growth substrates. Rather these compounds were metabolized by thestraintodetoxifyitsenvironmentandtheresulting metabolites have been occasionally used for growth up to the stationery phase in both biotransformations. The suggested pathways, however, are based solely on metabolic data. Therefore it is necessary to verify the presence of the postulated enzymes by protein biochemical or genetic analyses. As until now no other isolated acetylene hydratase has been described, a purification, sequencing, and protein biochemical characterization of the initial acetylene hydratase is inevitable. In case of the other, more common enzymes, which are involved a genomic sequence analysis and a comparison to the genome of other strains can also provide valuable information and enable protein identification. Apart from the hydrolyses investigated very little is known about microbial metabolism in other organisms that detoxify the environment from acetylenic compounds. To get a deeper insight into such biotransformations in situ 1 H-NMR analysis in 1 H 2 O is a valuable analytical method, although the substrates themselves are often ÔinvisibleÕ. Several metabolites can be detected, identified, and quantified directly from the cell culture or from the supernatant in concentrations > 0.2 m M . The use of natural 1 H 2 O excludes virtual reactions and does not affect growth rates by means of isotopic effects [16]. Addition of defined amounts of D 2 O, however, is useful to determine the incorporation of protons from the solvent into the products. Therefore this analytical technique allows a detailed analysis of the acetylene bond biodegradation in several organisms. Acknowledgements H. Griengl (Graz) and W. Steiner (Graz) are gratefully acknowledged for substantial contribution to this project. We thank G. Straganz (Graz) for valuable help and support performing the biochemical work. L. B. gratefully acknowledges Whiteknight Technologies, Ltd. (Exeter, GB) for financial support. References 1. Beilstein Crossfire, Version 3.1. Database PS0201PR. (1996–2002) Beilstein Information Systems GmbH, Frankfurt, Germany. 2. Benton, P.M.C., Christiansen, J., Dean, D.R. & Seefeldt, L.C. (2001) Stereospecificity of acetylene reduction catalyzed by nitrogenase. J. Am. Chem. Soc. 123, 1822–1827. Fig. 5. 1 H-NMR spectrum of 2-butyndioate metabolism, taken from the supernatant after 36 h. This shows the intermediate pyruvate, the main products acetate and formate, as well as the small amount of the by-product lactate. Fig. 6. 2-Butynedioate metabolic pathway in the isolated P. putida strain. The substrate was initially hydrolysed to 2-ketobutandioate and its appropriate enol isomer. This intermediate was decarboxylated to carbon dioxide and pyruvate. About 90% of the latter metabolite was than further hydrolysed to acetate and formate. Approximately 10% of the pyruvate was reduced to lactate, probably incorporating hydrogen from the formate, which was metabolized further. Ó FEBS 2003 1 H-NMR of a-alkynoate biotransformations (Eur. J. Biochem. 270) 1397 3. Rosner, B.M., Rainey, F.A., Kroppenstedt, R.M. & Schink, B. (1997) Acetylene degradation by new isolates of aerobic bacteria and comparison of acetylene hydratase enzymes. FEMS Micro- biol. Lett. 148, 175–180. 4. Walsh, C. (1977) Recent developments in suicide substrates and other active site-directed inactivating agents of specific target enzymes. Horizons Biochem. Biophys. 3, 36–81. 5. Marcotte, P. & Walsh, C. (1978) Sequence of reactions which follow enzymic oxidation of propargylglycine. Biochemistry 17, 5613–5619. 6. Miesowicz, F.M. & Bloch, K. (1979) Purification of hog liver isomerase. Mechanism of isomerization of 3-alkenyl and 3-alkynyl thioesters. J. Biol. Chem. 254, 5868–5877. 7. Ahmed, S. (1991) Microbial oxidative reactions of arenes 1 PhD Thesis. Imperial College, London, UK. 8. van den Tweel, W.J.J. & De Bont, J.A.M. (1985) Metabolism of 3-butyn-1-ol by Pseudomonas BB1. J. Gen. Microbiol. 131, 3155– 3162. 9. Yamada, E.W. & Jakoby, W.B. (1958) Enzymatic utilization of acetylenic compounds–I. An enzyme converting acetylenedicarb- oxylic acid to pyruvate. J. Biol. Chem. 233, 706–711. 10. Yamada, E.W. & Jakoby, W.B. (1958) Enzymatic utilization of acetylenic compounds – II. Acetylenemonocarboxylic acid hyd- rase. J. Biol. Chem. 233, 941–945. 11. Brecker, L. & Ribbons, D.W. (2000) Biotransformations moni- tored in situ by proton nuclear magnetic resonance spectroscopy. Trends Biotechnol. 18, 197–202. 12. Weber, H. & Brecker, L. (2000) Online NMR for monitoring biocatalysed reactions. Curr. Opin. Biotechnol. 11, 572–578. 13. Gillies, R.J. (1994) NMR in Physiology and Biomedicine.Academic Press, New York. 14. Barbotin, J.N. & Portais, J.C. (2000) NMR in Microbiology. Theory and Applications. Horizon Scientific Press, Wymondham, UK. 15. Field, L.D. & Sternhell, S. (1989) Analytical NMR. John Wiley & Sons, New York, USA. 16. Brecker, L., Weber, H., Griengl, H. & Ribbons, D.W. (1999) In situ proton-NMR analyses of Escherichia coli HB101 fermen- tations in 1 H 2 OandinD 2 O. Microbiology 145, 3389–3397. 17. Morawski, B., Eaton, R.W., Rossiter, J.T., Guoping, S., Griengl, H. & Ribbons, D.W. (1997) 2-Naphthoate catabolic pathway in Burkholderia strain. JT 1500. J. Bacteriol. 179, 115–121. 18. Gue ´ ron, M., Plateau, P. & Decorps, M. (1991) Solvent signal suppression in NMR. Prog. NMR Spectrosc. 23, 135–209. 19. Hore, J.P. (1989) Solvent suppression. In Methods in Enzymology (Oppenheimer, N.J. & James, J.T., eds), Vol. 176, pp. 64–77. Academic Press, Inc., San Diego, CA, USA. 1398 L. Brecker et al. (Eur. J. Biochem. 270) Ó FEBS 2003 . In situ proton NMR analysis of a-alkynoate biotransformations From ‘invisible’ substrates to detectable metabolites Lothar. deeper insight into such biotransformations in situ 1 H -NMR analysis in 1 H 2 O is a valuable analytical method, although the substrates themselves are often

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