Download Threonine Metabolism via Two-carbon Compounds

Journal of General Microbiology (1g71),67, 243-246
Printed in Great Britain
243
Threonine Metabolism via Two-carbon Compounds by
Pseudomonas oxalaticus
By M A U R E E N A. B L A C K M O R E A N D J. M. T U R N E R
Department of Biochemistry, University of Liverpool, Liverpool, L69 3BX
(Accepted for publication
I
June I 97 I )
A number of micro-organisms capable of growth on L-threonine degrade it via two-carbon
compounds. A species of Arthrobacter oxidized threonine by a dehydrogenase reaction to
2-amino-3-oxobutyrate which was then cleaved to acetyl-CoA plus glycine by a thiolase
reaction (McGilvray & Morris, 1969). In contrast, Pseudomonas putida split threonine directly
by an aldolase reaction to acetaldehyde plus glycine (Morris, 1969). In each case glycine was
metabolized by the folate-dependent serine pathway.
In the present study, Pseudomonas oxalaticus, originally isolated for its ability to grow
on oxalate, was concluded to catabolize threonine via acetyl-CoA plus glycine. Glycine
was metabolized via glyoxylate and the glycerate pathway, whilst acetyl-CoA was oxidized
via the tricarboxylic acid cycle.
RESULTS
Growth and manometric studies. Pseudomonas oxalaticus (NCIB8624) grew well at 30" on
basal media supplemented with L-threonine, or a variety of other compounds, as sole source
of carbon. Liquid media contained 7 g. K,HPO,, 3 g. KH2P04, 1-2 g. Na2S04, 0-1g.
MgS04.7Hz0 and 4 g. carbon source made up to I 1. with glass-distilled water. Where carbon
compounds contained no nitrogen, I g. (NH4)2S04was added. Growth on threonine was
not enhanced by NH+4ions, and growth on glycine alone did not occur unless the inoculum
had been grown initially on threonine medium. Good growth occurred on acetate alone.
Growth on an equimolar mixture of acetate plus glycine took place without lag as rapidly
as growth on threonine. No growth occurred on 2-oxobutyrate medium.
Washed suspensions of Pse udomonas oxalaticus grown on L-threonine oxidized a mixture of
acetate + glycine more rapidly than either compounds used singly or indeed threonine itself.
Relative rates, measured manometrically in Warburg flasks using conventional procedures
at 30°, were : L-threonine, 100; acetate, 43; glycine, 46; acetate+glycine, 120; L-serine, 103;
glyoxylate , 67 ;glycolate, 50; DL-lactate, 103; and pyruvate, 109. 2-Oxobutyrate was oxidized
only slowly, and aminoacetone, DL-I-aminopropan-2-01and methylglyoxal not at all. When
grown on succinate the micro-organisms could no longer oxidize L-threonine or glyoxylate.
Enzymes utilizing L-threonine. The activities of L-threonine dehydrogenase, dehydratase
and aldolase were measured in crude extracts of Pseudomonas oxalaticus grown on either
L-threonine, acetate or succinate a sole carbon source. The results are shown in Table I .
No aldolase activity co uld be detected. Threonine dehydratase activity in all extracts was
markedly inhibited by isoleucine. Some properties of the inducibly formed L-threonine
dehydrogenase were determined after partial purification by molecular exclusion chromatography on a column of BioGel P-300. Optimum activity was at pH 9.0 to 9-5, and kinetic
constants determined by the graphical procedure of Lineweaver & Birk (1934) were: K ,
L-threonine = 8.0 mM; K , NAD+ = 0.16 mM; and V,,, = 1-35 units/rng. protein. Only
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Tue, 01 Aug 2017 23:51:04
Short commun ication
244
30 % activation was found with KC1 at 0.5 M. Inhibition was observed with HgCl,, 100 yo
at I mM; p-hydroxymercuribenzoate, 82 yo at I m; 1,Io-phenanthroline, 55 Yo at I mM;
and 2,2'-bipyridyl, 36% at I mM. Iodoacetate and EDTA had no effect at 5 mM. Additions
were preincubated with enzyme in buffer for 15 min. before activity assay.
Aminoacetone formation from L-threonine. Washed suspensions of threonine-grown
Pseudomonas oxalaticus did not accumulate aminoacetone when incubated with the amino
acid. During growth on L-threonine only low concentrations (less than 0.05 mM) accumulated
during the lag phase. Slow but complete disappearance of aminoacetone occurred when
rapid growth commenced. When iodoacetate (25 mM) was added to growing cultures,
aminoacetone accumulated up to concentrations of about 0.50 mM within 24 h.
Table
I.
Enzyme activities in extracts of Pseudomonas oxalaticus grown on various
sources of carbon
The micro-organism was grown as described in the text, harvested by centrifugation (20,000 g
for 10min.) and disrupted ultrasonically. L-Threonine dehydrogenase was measured at 37" by
the colorimetric method of Mauzerall & Granick (I 956) as previously described (Turner, I 966).
L-Threonine dehydratase activity was assayed at 37" by the method of Ning & Gest (1966). Aldehyde
dehydrogenase was measured spectrophotometrically as described by King & Cheldelin (1 956),
glyoxylate carboligase manometrically (Blackmore & Quayle, I 968) and glyoxylate aminotransferase (Rowsell, Snell, Carnie & Al-Tai, I 969) and isocitrate lyase spectrophotometrically (Dixon,
& Kornberg, 1959). Enzyme activities were measured at 30" unless otherwise stated. Units of
enzyme are defined as those amounts which catalysethe formation of I pmole of product (or transformation of substrate)/min.under the conditions described.
Enzyme specific activities (units/mg. protein)
A
r
Growth
substrate
L-Threonine
Acetate
Succinate
L-Threonine
dehydro- L-Threonine
genase
dehydratase
(37")
(37")
0.720
0'01I
0.040
0.025
0.016
0.050
l
L-Alanine
Aldehyde
dehydrogenase
Glyoxylate
carboligase
glyoxylate
amino
transferase
Isocitrate
lyase
0.038
0.070
0.036
0-008
0'012
0'120
0.024
0.037
0.006
0
0.0I 8
0
Suspensions of Pseudomonas oxalaticus grown on L-threonine did not remove aminoacetone from suspension media. Cell-free extracts were unable to catalyse the 2-0x0 acid
and NAD+-dependent utilization of aminoacetone observed with Pseudomonas and Bacillus
species (Higgins, Turner & Willetts, 1967).
2-Amirzo-3-oxobutyrate CoA ligase. This enzyme, originally considered to be 'aminoacetone synthase', was implicated in the catabolism of threonine to acetyl-CoA plus glycine
by McGilvray & Morris (I 969). Activity in extracts of threonine-grown Pseudomonas oxalaticus
could not be measured satisfactorily by the methods used by these authors due to the presence
of an active acylase (approx. 150 nmoles/mg. protein/min. at 30") present under all conditions tested. The cosubstrate was hydrolysed so rapidly that neither CoA nor aminoacetone
formation from acetyl-CoA plus glycine could be detected.
Metabolism of acetate and glycine by Pseudomonas oxalaticus. When the threonine-grown
micro-organism was resuspended in either acetate or glycine growth media, growth occurred
after a lag of 4 to 5 h. There was no lag in acetate+glycine medium. Threonine-grown
micro-organisms oxidized an equimolar mixture of acetate and glycine more rapidly than
threonine itself.
Enzymic evidence suggested that glycine was metabolized via glyoxylate and the glycerate
pathway (Dagley, Trudgill & Callely, 1961;Kornberg & Gotto, 1961). High glycine-pyruvate
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Tue, 01 Aug 2017 23:51:04
Short communication
245
aminotransferase and glyoxylate carboligase activities were found in extracts of Pseudomonas
oxalaticus grown on threonine in contrast to succinate or acetate media (see Table I). No
other transaminations involving glyoxylate and a variety of amino acids including L-serine
(Blackmore & Quayle, 1970) and L-aspartate (R. G. Gibbs & J. G. Morris, personal communication) could be detected. P-Hydroxyaspartate dehydratase activity (Kornberg & Morris,
1965) could not be detected. Low isocitrate lyase activity (Dixon & Kornberg, 1959) was
found after growth on threonine rather than acetate (see Table I). Malate and citrate synthase activities could not be assayed satisfactorily, again because of high acylase activity in
crude extracts.
D I S C US S I O N
Growth, manometric and enzymic studies all suggested that L-threonine was catabolized
by Pseudomonas oxalaticus via acetyl-CoA glycine rather than aminoacetone. Threoninegrown bacteria adapted to growth on acetate+glycine medium without a lag and rapidly
oxidized an equimolar mixture of these compounds. It appeared likely that acetyl-CoA was
oxidized via the TCA cycle and that glycine was metabolized by a pathway functioning
anaplerotically.
L-Threonine dehydrogenase formation induced by growth on threonine indicated that the
initial step of threonine catabolism was the formation of 2-amino-3-oxobutyrate. The specific
activity of the enzyme in extracts was higher than that previously described from any source.
Extensive purification and partial resolution of the enzyme into two components has
recently been achieved in these laboratories (Lowe & Dean, 1971). The absence of L-threonine
aldolase, and aldehyde dehydrogenase activity unaffected by growth on threonine, ruled
out the aldolytic cleavage of threonine observed in other Pseudomonas strains (Morris,
I 969). Low threonine dehydratase activity markedly inhibited by isoleucine, suggesting a
biosynthetic rather than catabolic role, and the inability of Pseudomonas oxalaticus to
grow on 2-oxobutryate or oxidize this compound, eliminated deamination as the initial
obligatory catabolic step.
Although 2-amino-3-oxobutyrate thiolase could not be detected, probably for technical
reasons, the accumulation of aminoacetone when iodoacetate was added to cultures of
Pseudomonas oxalaticus growing on threonine was consistent with the catabolism of the
3-0x0 acid by a CoA-dependent step. The failure of threonine-grown bacteria to oxidize or
otherwise utilize aminoacetone and oxidize methylglyoxal, and the absence of ‘aminoacetone
aminotransferase activity ’ (Higgins, Turner & Willetts, I 967) from extracts precluded the
operation of the amino ketone pathway (Willetts & Turner 1970) and contrasts with the
properties of a Pseudomonas strain grown on I -aminopropan-2-01 (Higgins, Pickard &
Turner, 1968).
The ability of the threonine-grown bacteria to oxidize glycine and glyoxylate, in contrast
to P-hydroxyaspartate, together with the finding of glycine-pyruvate aminotransferase and
glyoxylate carboligase activities in extracts, indicated the operation of the glycerate pathway
(Kornberg & Elsden, 1961).It is known that Pseudomonas oxalaticus uses the glycerate pathway for glyoxylate metabolism during growth on oxalate (Blackmore & Quayle, 1970).
P-Hydroxyaspartate dehydratase activity (Kornberg & Morris, I 965) could not be detected.
Glyoxylate carboligase formation was markedly induced by growth on threonine (Table I).
The finding of low isocitrate lyase activity after growth on threonine rather than acetate was
additional evidence that glycine was metabolized via the glycerate pathway functioning
anaplerotically .
+
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Tue, 01 Aug 2017 23:51:04
246
Short comrnunicat ion
The interest and support of Professor T. W. Goodwin, F.R.S., is gratefully acknowledged.
The authors also thank Miss E. Duggan for expert technical assistance.
REFERENCES
BLACKMORE,
M. A. & QUAYLE,
J. R. (1968). Choice between autotrophy and heterotrophy in Pseudomonas
oxalaticus : growth in mixed substrates. Biochemical Journal 107,705-71 3.
BLACKMORE,
M. A. & QUAYLE,
J. R. (1970). Microbial growth on oxalate by a route not involving glyoxylate
carboligase. Biochemical Journal 118,53-59.
A. G. (1961). Synthesis of cell constituents by a Pseudomonas.
DAGLEY,S.,TRUDGILL,P. W. & CALLEY,
Biochemical Journal 81,623-631.
H. L. (1959).Assay methods for key enzymes of the glyoxylate cycle. Biochemical
DIXON,G, H. & KORNBERG,
Journal 72,3P.
HIGGTNS,
I. J., PICKARD,
M. A. & TURNER,
J. M. (1968). Aminoacetone formation and utilization by pseudomonads grown on DL-I-aminopropan-2-01.Journal of General Microbiology 54, 105-1 14.
A. J. (1967). Enzyme mechanism of aminoacetone metabolism
HIGGINS,I. J., TURNER,J. M. & WILLETTS,
by micro-organisms. Nature, London 216,887-888.
KING,T. E. & CHELDELIN,
V. H, (1956). Oxidation of acetaldehyde by Acetobacter suboxydans. Journal of
Biological Chemistry 220, I 77-191.
H. L. & ELSDEN,S. R. (1961). The metabolism of two-carbon compounds by micro-organisms
KORNBERG,
Advances in Enzymology 23, 401-470.
KORNBERG,
H. L. & GOTTO,A. M. (1961). The metabolism of C, compounds in micro-organisms. Synthesis
of cell constituents from glycollate by Pseudomonas sp. Biochemical Journal 78,69-82.
KORNBERG,
H.L. & MORRIS,J. G. (1965). The utilization of glycollate by Micrococcus denitrificans: the
P-hydroxyaspartate pathway. Biochemical Journal 95,577-586.
LINEWEAVER,
H.& BURK,D. (1934). The determination of enzyme dissociation constants. Journal of the
American Chemical Society 56, 658-663.
L o w , C. R. & DEAN,P.D. G. (1971). Affinity chromatography of enzymes on insolubilized cofactors.
FEBS Letters 14,3 13-3 I 6.
MAUZERALL,
D. & GRANICK,S. (1956). The occurrence and determination of baminolevulinic acid and
porphobilinogen in urine. Journal of Biological Chemistry 219,435-446.
MCGILVRAY,
D. (I 967). The assimilation of L-threonine by an Arthrobacter species. Ph.D. Thesis, University
of Leicester.
MCGILVRAY,
D. & MORRIS,J. G. (1969). Utilization of L-threonine by a species of Arthrobacter: A novel
catabolic role for ‘aminoacetone synthase’. Biochemical Journal 112, 657-671.
NING,C. & GEST,H. (1966). Regulation of isoleucine biosynthesis in the photosynthetic bacterium Rhodospirillum rubrum. Proceedings of the National Academy of Sciences of the United States of America 56,
I 823-1 827.
MORRIS,J. G. (1969). Utilization of L-threonine by a pseudomonad: a catabolic role for L-threonine aldolase.
Biochemical Journal 115, 603-605.
ROWSELL,
E. V., SNELL,K., CARNIE,
J. A. & AL-TAI,A. H. (1969). Liver L-alanine-glyoxylate and L-serinepyruvate aminotransferase : an apparent association with gluconeogenesis. Biochemical Journal 115,
1071-1073.
TURNER,J. M. (I 966). Microbial metabolism of aminoketones. Aminoacetone formation from I -aminopropan-2-01 by a dehydrogenase in Escherichia coli. Biochemical Journal 99,427-433.
WILLETTS,
A. J. & TURNER,
J. M. (1970). Threonine metabolism in a strain of Bacillus subtilis. Biochemical
Journal ~17,
27-28~.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Tue, 01 Aug 2017 23:51:04