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

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

[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

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

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

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

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

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,

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

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
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Academic Press, Inc., San Diego, CA, USA.
1398 L. Brecker et al. (Eur. J. Biochem. 270) Ó FEBS 2003