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Journal of Bacteriology, September 2001, p. 5203-5205, Vol. 183, No. 17
Department of Biochemistry, Virginia
Polytechnic Institute and State University, Blacksburg, Virginia
24061-0308
Received 26 March 2001/Accepted 7 June 2001
Cell extracts of Methanococcus jannaschii have been
shown to readily convert L-ornithine to
L-proline. This cyclization reaction proceeds with the loss
of only the C-2 nitrogen, as has been documented for ornithine
cyclodeaminase (EC 4.3.1.12). Since no gene homologous to that coding
for ornithine cyclodeaminase is present in the genome of M.
jannaschii, these results indicate that proline biosynthesis in
M. jannaschii is accomplished by a previously
unrecognized enzyme.
The established pathway for proline
biosynthesis in microorganisms is shown in the upper portion of Fig.
1. The reaction sequence involves (i) the
phosphorylation of the
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.17.5203-5205.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Methanococcus jannaschii Generates
L-Proline by Cyclization of
L-Ornithine
ABSTRACT
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-carboxyl of L-glutamate to form
L-glutamyl-5-P, (ii) the NADH-dependent reduction of
L-glutamyl-5-P to glutamic acid-
-semialdehyde, (iii) the
cyclization of glutamate--semialdehyde to
1-pyrroline-5-carboxylic acid, and (iv) the
reduction of 1-pyrroline-5-carboxylic acid to
L-proline (2, 5, 13). As first pointed out by
Selkov et al. for Methanococcus jannaschii (10), and later as a general characteristic of the genomes
of most of the Archaea, genes coding for the three enzymes
in this pathway are largely absent in the Archaea
(4). In contrast, the genes for the biosynthesis of
L-ornithine are generally present in all of the
archaeal genomes (4). A simple solution to explain the
absence of the proline biosynthetic genes in the genomes of some of the
Archaea is that proline is derived by the cyclization of
L-ornithine. This reaction could be accomplished
by ornithine cyclodeaminase (EC 4.3.1.12), an enzyme which is presently
considered to have a limited distribution among the bacteria
(12). Ornithine cyclodeaminase was first isolated from
Clostridium sporogenes, where it functions as the first
step in the anaerobic catabolism of
L-ornithine via proline to -aminovaleric acid
(1). The proposed chemical steps for the mechanism of this
enzyme involve the oxidative deamination of the 
-amino group of
ornithine to 2-oxo-5-aminopentanoic acid, which cyclizes to
1-pyrroline-2-carboxylic acid, which is
subsequently reduced to L-proline. The ornithine
cyclodeaminase has been shown to contain 1 mol equivalent of bound
NAD+, which is considered to function as a
recycling redox carrier in this transformation (7). Mass
spectroscopic data showed that [5-15N]ornithine
is converted to [15N]proline by ornithine
cyclodeaminase, confirming that the initial oxidation of the ornithine
is at C-2 (8). Using deuterated ornithine and
[15N]ornithine, we have now demonstrated that
proline in M. jannaschii is derived from ornithine by a
mechanism that is analogous to that demonstrated by the ornithine
cyclodeaminase. Since there is no enzyme-encoding gene with a sequence
homologous to that coding for the ornithine cyclodeaminase present in
the M. jannaschii genome, we propose that a currently
unidentified enzyme, functioning with an analogous mechanism, is
involved in proline biosynthesis in M. jannaschii.
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FIG. 1.
Established and proposed pathways for proline
biosynthesis.
Preparation and analysis of cell extracts.
Cell extracts of
M. jannaschii, Methanosarcina thermophila strain
TM-1, and Methanobacterium thermoautotrophicum strains H and Marburg were prepared as previously described (16).
The protein concentrations of the cell extracts used typically
ranged from 7 to 26 mg/ml.
Incubation with substrates.
Cell extracts (50 µl) were
incubated with millimolar concentrations of the substrates under argon
for 2 h at 50°C.
L-[2,4,4'-2H3]glutamic
acid and
L-[3,3',4,4',5'-2H6]ornithine
were obtained from Cambridge Isotope Laboratories, Inc.
[5-15N]ornithine was prepared from potassium
[15N]-phthalimide by the following
series of reactions. Potassium [15N]-phthalimide (98 atom%
15N) was reacted with dibromopropane in acetone
to form 15N-labeled
N-(3-bromopropyl)phthalimide (14), which was
condensed with ethyl acetamidocyanoacetate in ethanol in the presence
of sodium ethoxide. Acid hydrolysis of the condensation product (6 M
HCl, 24 h, 110°C) and separation of the resulting products on a
Dowex 50-8X (H+) column with an HCl gradient
resulted in the isolation of chromatographically pure
[5-15N]ornithine.
1-Pyrroline-5-carboxylic acid was prepared
from the 2,4-dinitrophenylhydrazine derivative as previously described
(6).
-glutamate present in
the M. jannaschii cell extracts (9) was used as
an internal standard. The establishment of the product of the
incubation as L-proline was accomplished by GC-MS
of the methyl ester trifluoroacetyl derivative using a type G-TA
Chiraldex column as previously described (3).
As can be seen from the data presented in Table
1, incubation of a cell extract of
M. jannaschii with
[2,4,4'-2H3]glutamic
acid, ATP, NADH, and NADPH produced no detectable amount of labeled
proline. Likewise, incubation with
1-pyrroline-5-carboxylic acid, NADH, and NADPH
at the same concentrations failed to produce any detectable amount of
proline (data not shown). Incubation of the cell extract with
L-ornithine (9.1 mM) resulted in the production
of 0.13 µmol of proline, which corresponded to the conversion of 26%
of ornithine to proline. To confirm that ornithine was the sole
precursor of the proline, we incubated the cell extracts with
L-[3,3',4,4',5,5'-2H6]ornithine
and measured the incorporation of six deuteriums in the generated
proline, which indicated that the carbon skeleton of the proline was
derived from the ornithine as an intact unit. To establish which
of the nitrogens was lost in the cyclization, the experiment was
repeated with [5-15N]ornithine, and the
recovered proline contained 98% 15N. This result
showed that the C-2 nitrogen was the one lost in the cyclization. Since
the direct displacement of the C-2 amino group by the C-5 amino group
is without enzymatic or chemical precedent, the most likely chemical
steps for the reaction would involve the oxidation of the C-2 carbon to
an imine, intramolecular cyclic addition of the C-5 nitrogen to the C-2
carbon, loss of ammonia, and reduction of the resulting imine,
1-pyrroline-2-carboxylic acid, as shown in
lower portion of Fig. 1. Alternately, the imine intermediate generated
in the first oxidation could undergo hydrolysis to the keto acid before
the cyclization would occur. In this case, elimination of water would produce the 1-pyrroline-2-carboxylic acid.
Although not directly confirmed by the data, the likely choice for the
coenzyme to be involved in this process would be an enzyme-bound
NAD+ as occurs in ornithine cyclohydrolase. The
possible involvement of coenzyme F420 must also
be considered since coenzyme F420 can also effect
hydride transfer reactions (15).
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H and strain Marburg, indicating
that these autotrophs generate their proline by the same mechanism as
that found in M. jannaschii. Although no gene coding for
ornithine cyclodeaminase can be found in the M. jannaschii
genome, a gene homologous to one coding for C. sporogenes
ornithine cyclodeaminase is present in the M. thermoautotrophicum H (11) and
Methanosarcina barkeri (http://www.jgi.doe.gov)
genomes. Cell extracts of M. thermophila strain TM-1, on the
other hand, were found to produce no proline from ornithine. The
M. barkeri genome also has the genes for the biosynthesis of
proline from glutamate via
1-pyrroline-5-carboxylic acid. These genomic
data indicate that several different routes may be operating in the
Archaea for the biosynthesis of proline. Although our data
cannot rule out the possibility that additional pathways to proline
function in vivo, this seems unlikely due to the efficient conversions
from ornithine that were observed in vitro.
In total, these observations show that an enzyme with no homology to
the known ornithine cyclodeaminases is involved in proline biosynthesis
in M. jannaschii. We are presently in the process of
isolating the enzyme responsible for carrying out the reaction demonstrated here in order to establish the gene required for its production.
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ACKNOWLEDGMENTS |
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We thank Kim Harich for the GC-MS analyses and David Graham for
reviewing the manuscript prior to publication. We thank James G. Ferry
for the cells of M. thermophila strain TM-1, Ralph S. Wolfe for M. thermoautotrophicum strains H and
Marburg, and Biswarup Mukhopadhyay for cells of M.
jannaschii.
This work was supported in part by National Science Foundation grant MCB9985712.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0308. Phone: (540) 231-6605. Fax: (540) 231-9070. E-mail: rhwhite{at}vt.edu.
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Journal of Bacteriology, September 2001, p. 5203-5205, Vol. 183, No. 17
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.17.5203-5205.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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