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 Pseudomona
Trang 1In situ proton NMR analysis of a-alkynoate biotransformations
From ‘invisible’ substrates to detectable metabolites
Lothar Brecker1,*, Julia Petschnigg1, Nicole Depine´1, Hansjo¨rg Weber1and Douglas W Ribbons1,2,†
1
Institute of Organic Chemistry, University of Technology Graz, Austria;2Institute 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-hydroxypro3-ketopro-panoate Analyses of these meta-bolic processes were performed by in situ 1H-NMR spectroscopy in1H2O, 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 situ1H-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 (1H-NMR) in water (1H2O) as
a versatile analytical method [11–15] This technique allows
1H-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 invisible in 1H-NMR The incorporation of protons from the solvent in the pathway, however, makes the metabolites detectable Therefore additional defined amounts of deuterium (D2O) in water (1H2O) can easily
be detected in the metabolites and allow conclusions about details of the pathway [16] Here we describe the in situ
1H-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)
Trang 2plates [17] containing the substrates (5 mM) at pH 7.
Liquid cultures were grown on a rotary shaker at 30°C
and pH 7 in mineral medium [17] supplemented with
5 mM 2-butynedioate or 5 mM propynoate, respectively
Samples were taken every hour for determination of the
optical density (D570) After 24 h cells were centrifuged,
washed, and resuspended in 100 mM phosphate buffer in
1H2O (pH 7) or 1H2O/D2O (1 : 1, pH 7) to a
suspen-sion with a D570 of 0.3–0.5 Aerobic biotransformations
to determine the metabolism time courses at 30°C
were started by addition of the substrate (5 mM) to
these suspensions They were sampled at every 2 h for
UV measurements and then every 12 h for 1H-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 (D570) 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 1H-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 D2O vortex capillary was added to the
NMR tube to avoid 1H/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; 1H 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-d4 acid (TSP) signal (1H d: 0.00 p.p.m) was used
as an external reference For in situ 1H-NMR
measure-ment we analysed the supernatant of the cell cultures All
metabolites were identified by addition of standard
solutions to the sample
(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
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].
Trang 3pathways of the two acetylenic compounds UV spectroscopy
and in situ1H-NMR [11–15] were used
Biotransformation of propynoate
The metabolism of propynoate (kmax¼ 263 nm) was first
analysed by UV indicating that the substrate concentration
(5 mM) 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 1H-NMR analysis (1H
d: 3.06 p.p.m) in 1H2O Spectra indicated the additional
presence of a small amount of 3-ketopropanoate (1H
d: 3.48 p.p.m) and acetaldehyde (1H 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 mM(Fig 3a and b) In parallel the amount of acetate
formed (1H d: 1.90 p.p.m) and 3-hydroxypropanoate (1H
d: 2.42 p.p.m) increased up to 2.5 mMwhile 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 mMand 5 mM) to the biotransformation The 0.5 mM 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
5 mMacetaldehyde obviously induced other enzymes that metabolise this substrate in a different way
Performing the propynoate biotransformation in1H2O/
D2O (1 : 1) led to an incorporation of 50% deuterium in all metabolites The addition of 0.5 mM acetaldehyde to this biotransformation in 50% D2O caused an 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 is shown in Fig 4
Biotransformation of 2-butynedioate The UV spectrophotometric analysis of the 2-butynedioate metabolism provided scattered absorptions at kmax¼
265 nm during the first 24 h of the biotransformation Furthermore the consumption of 2-butynedioate could not
be monitored by1H-NMR due to the lack of protons in this substrate However, 1H-NMR clearly indicates the formation of pyruvic acid [1H 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.
Trang 41.43 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 (1H d: 1.87 p.p.m) and formate (1H 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 (1H 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.
Trang 5resulting 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
the strain to detoxify its env ironment and the resulting
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 1H-NMR analysis in1H2O is a valuable analytical method, although the substrates themselves are often invisible Several metabolites can be detected, identified, and quantified directly from the cell culture or from the supernatant in concentrations > 0.2 mM The use of natural1H2O excludes virtual reactions and does not affect growth rates by means of isotopic effects [16] Addition of defined amounts of D2O, 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.
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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.
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