Download Specific Activities of Enzymes of the Serine Pathway of Carbon

542nd MEETING, SOUTHAMPTON
1291
Specilk Activities of Enzymes of the Serine Pathway of Carbon
Assimilation in Pseudomonas aminovorans and Pseudomonas MS
grown on Methylamine
PETER J. LARGE and ROBERT H. CARTER
Department of Biochemistry, University of Hull, Hull HU6 7RX, U.K.
Very little is known about the pathway of carbon assimilation during growth on C,
compounds of Pseudomonas aminovorans, a facultative methylotroph that will grow on
methylamine, dimethylamine, trimethylamine or trimethylamine N-oxide as sole carbon
source, although the pathways by which the amines are oxidized are being studied (Large,
1971). We decided to examine extracts of bacteria grown on methylamine for enzymes of
the serine pathway of carbon assimilation (Quayle, 1972) and to compare the specific
activities with corresponding values for cells grown on succinate, where these enzymes
would have either no role or a quantitatively less important role in growth. Wagner &
QuayIe (1972) have suggested that Pseudomonas MS, a similar facultative methylotroph,
does not use the serine pathway for growth on methylamine. They based this conclusion
on two observations: (a) that the first labelled product of [14C]methylamineassimilation
was N-methylglutamate, not serine, and (b)that they were unable to detect hydroxypyruvate reductase (glycerate dehydrogenase) in extracts of methylamine-grown Pseudomonas
MS. I n view of evidence (Bellion & Hersh, 1972) that N-methylglutamate is an intermediate in the oxidation rather than the assimilation of methylamine (a view for which
we have evidence in Ps. aminovorans also), we decided to examine enzyme specific activities in extracts of methylamine- and succinate-grown Pseudomonas MS at the same
time.
The pH-dependence of the enzymes hydroxypyruvate reductase, phosphoenolpyruvate
carboxylase and serine-glyoxylate amhotransferase were examined in Ps. aminovorans,
and the optima were respectively pH6.0, 7.0 and 7.5. Table 1 shows that both
organisms when grown on methylamine possessed elevated activities of the enzymes
serine hydroxymethyltransferase, hydroxypyruvate reductase, malate dehydrogenase,
phosphoenolpyruvate carboxylase, serineglyoxylate aminotransferase and ATP malate
lyase (Hersh & Bellion, 1972). The ratio of specific activities (methylamine-grown cells/
succinate-grown cells) was above 40 in each case. These enzymes can account for the net
conversion of a reduced C1 unit and COz into acetyl-CoA via serine. The results also
suggest that serine dehydratase is not involved in this sequence of reactions. The ATP
malate lyase activity detected was not very high, compared with that in other amineutilizing organisms (Bellion & Hersh, 1972; Cox & Zatman, 1973), but was absolutely
dependent on CoA. ATP and Mg2+,although the identity of glyoxylate as reaction product was not conclusively established. We have not yet looked for a malyl-CoA-cleavage
enzyme, but its presence is a possibility in view of the observations made by Salem et al.
(1973). The evidence supports the concept of a cyclic mechanism for the generation of
acetyl-CoA in these two organisms from formaldehyde (produced by oxidation of
methylamine via N-methylglutamate) and COz via serine and hydroxypyruvate, as
postulated for Pseudomonas AM1 (Salem et al., 1972) and bacterium 5H2 (Cox &
Zatman, 1973), with the glyoxylate required for the cycle probably arising via cleavage of
malate.
We propose that in both Pseudomonas MS and Ps. aminovorans carbon is assimilated
via the serine pathway, the route from methylamine to C, acids being similar to that
proposed by Bellion & Hersh (1972) for Pseudornonas MA, but proceeding from serine
via glycerate and phosphoenolpyruvate (Large & Quayle, 1963), as in Scheme 1, rather
than involving serine dehydratase and ‘malic’ enzyme. The failure of Wagner & Quayle
(1972) to detect hydroxypyruvatereductase is due to their performing the assay at pH4.5.
At this pH we also were unable to detect activity, but very high activities were apparent
at pH5.5-6.0. Our failure to detect serine hydroxymethyltransferase in succinate-grown
bacteria, where it is presumably required for glycine biosynthesis (Harder & Quayle,
Vol. 1
ymethyltransferase (EC 2.1.2.1)
yruvate carboxylase
1)
rogenase (EC 1.1.1.27)
rogenase (EC 1.1.1.37)
yase (EC 4.1.3.-)
atase (EC 4.2.1.I 3)
e (EC 4.1.3.1)
Enzyme
ate reductase (glycerate
ase) (EC 1.1.1.29)
late aminotransferase
Bellion & Hersh (1972)
Kornberg (1955)
Scrimgeour & Huennekens (1962)
Large et al. (1962)
Blackmore & Quayle (1970)
Assay method
Large & Quayle (1 963)
0.2
340
125
100
2.3
442
0
16.6
5.5
0
0
0
Grown on
succinate
113 .
PS.aminovorans
Grown on
methylamine
4640
I
0.3
1170
8.1
2.4
10.6
320
157
1.3
314
0
22
1.3
0.8
27
4150
127
Grown on
succinate
Grown on
methylamine
Pseiidomonas MS
arison of speci3c activities of enzymes of the serinepathway in extracts ofPs. aminovorans andPseudomonas MSgrown on methylamine
te
Specific activity (nmol/min per mg of protein)
cl
r:
3
E
0
v)
R
En
Y
542nd MEETING, SOUTHAMPTON
Hydroxypyruvate
c1u
-
1293
Glycerate + Phosphoglycerate
n i t < L r H
Glyoxylate
d
I
21
Acetyl-CoA----.,
-
Oxaloacetate A C O ,
/
/
,
/'
v
\ I
\
\
,R
I
Malate
1
Phosphoenolpyruvate
'. Isocitrate +------- Citrate
A
I
J.
Succinate
Scheme 1. Proposedpathway of C,assimilation in Ps. aminovorans andpseudomonas MS
1971), may be due to theactivities being too low for detection by the relatively insensitive
assay method used (Scrimgeour & Huennekens, 1962).
The possibility that the acetyl-CoA formed by this pathway may condense with oxaloacetate to yield citrate and isocitrate followed by cleavage to succinate and glyoxylate, as
inpseudomonas MA and bacterium 5H2 (Bellion & Hersh, 1972; Cox & Zatman, 1973),
has also been investigated, but the relatively small (twofold) difference in specificactivity
of isocitrate lyase in extracts ofPseudomonas aminovoransgrown on methylamine and on
succinate does not allow a firm conclusion to be drawn, and this pathway is indicated by
broken lines in Scheme 1.
We thank Professor J. R. Quayle for the gift of a culture of Pseudomonas MS.
Bellion, E. & Hersh, L. B. (1972) Arch. Biochem. Biophys. 153, 368-374
Blackmore, M. A. & Quayle, J. R. (1970) Biochem. J. 118,53-59
Cox, R. B. & Zatman, L. J. (1973) Biochem. SOC.Trans. 1, 669-671
Harder, W. & Quayle, J. R. (1971) Biochem. J. 121, 753-762
Hersh, L. B. & Bellion, E. (1972) Biochem. Biophys. Res. Commun. 48, 712-719
Kornberg, A. (1955) Methods Enzymol. 1, 441443
Large, P. J. (1971) Xenobiotica 1,457467
Large, P. J. & Quayle, J. R. (1963) Biochem. J. 87, 386-396
Large, P. J., Peel, D. &. Quayle, J. R. (1962) 85, 243-250
Quayle, J. R. (1972) Aduan. Microbial Physiol. 7, 119-203
Salem, A. R., Large, P. J. & Quayle, J. R. (1972) Biochem. J . 128, 1203-1211
Salem, A. R., Hacking, A. J. & Quayle, J. R. (1973) J . Gen. Microbiol. 77, xii
Scrimgeour, K. G. & Huemekens, F. M. (1962) Methods Enzymol. 5, 838-843
Wagner, C. & Quayle, J. R. (1972) J. Gen. Microbiol. 72, 485-491
Dextransucrase from Streptococcus sanguis 804 : Characterization of
the Products
JOSIE A. BEELEY and PHYLLIS M. AYRES
Departments of Biochemistry and Oral Biology, University of Glasgow,
Glasgow G12 SQQ, U.K.
Dextransucrase [c+(l-+6)-glucan-~-fructose 2-glucosyltransferase, EC 2.4.1.51 is a
constitutive extracellular enzyme synthesized by Streptococcus sanguis 804, N.C.T.C.
10904 (Hehre & Neill, 1946; Carlsson et al., 1969), that polymerizes the glucose moiety
of sucrose to form dextran (Eisenberg & Hestrin, 1963).
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