3. RESULTS
3.1 Malate dehydrogenase and alanine dehydrogenase assay
Malate dehydrogenase and alanine dehydrogenase are enzymes which have been thoroughly
studied and characterized. Studies have proved that genes which encode malate
dehydrogenase and alanine dehydrogenase are present in RLV3841 and Bacteroids. These
assays were done to confirm the induction of these enzymes in RLV3841 grown on different
carbon source with ammonia and in bacteroids.
From the assay done, there was detectable activity of malate dehydrogenase in all RLV 3841
and bacteroids and the specific rate of activity obtained was consistent with previous studies
which are in the range of 1.0 – 5.0 µmole/min/mg protein (McKay et al., 1989). As for alanine
dehydrogenase, activity was also detected in all RLV 3841 and Bacteroids and the specific rate
of activity obtained is also within the range of previous studies which are in the range of 0.07 –
0.1 µmole/min/mg protein (Emma et al., 2004).
9
µmole/min/mg protein
8
7
6
5
4
3
2
1
0
3841 (Glucose)
3841 (Pyruvate)
3841 (Succinate)
Bacteroids
Figure 1: Comparison of rate of specific activity of malate dehydrogenase in free-living
Rhizobium leguminosarum bv. viciae strain 3841 grown on different carbon source and
ammonia and bacteroids.
0.16
µmole/min/mg protein
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
3841 (Glucose)
3841 (Pyruvate)
3841 (Succinate)
Bacteroids
Figure 2: Comparison of rate of specific activity of alanine dehydrogenase in free-living
Rhizobium leguminosarum bv. viciae strain 3841 grown on different carbon source and
ammonia and Bacteroids.
3.2 Other keto/hydroxy acid Assay
Metabolomics data (Unpublished data from Phil Poole research group) provided interesting
information on the different types of metabolites which were detected in RLV 3841 and
Bacteroids. Further enzyme assay was done using different metabolites of keto/hydroxy acids
which were detected in both RLV 3841 and bacteroids. All the keto/hydroxy acids were assayed
using different co-substrates (NADH, NAD+, NADP+ and NADPH) to see any activity which
might be induced and if the reaction was co-substrate specific. Particular interest was on the
induction of activity in bacteroids as some of the metabolites used in these assays were
detected more than 20 x up compared to RLV 3841 from the metabolomics data.
0.12
µmole/min/mg protein
0.1
0.08
0.06
RLV 3841 (Glucose)
RLV 3841 (Pyruvate)
0.04
RLV 3841 (Succinate)
Bacteroids
0.02
0
Figure 3: Enzyme specific rate activity when using different substrates of keto/hydroxy acids
with NADH as a co-substrate in free living Rhizobium leguminosarum bv. viciae strain 3841 and
Bacteroids.
The data from figure 3, shows a large error bars for the specific rate of activity, this is due to the
number of replicates of the assay being less than three. Ignoring the huge error bars, it can be
seen that the assay of keto/hydroxy acid with co-substrate NADH showed induction of enzyme
activity in all keto/hydroxy acid assay in all RLV 3841 and bacteroids. With the exception of
glycolic acid and glyoxylic acid assay in bacteroids. The non-induction for the glyoxylic acid in
bacteroids is linked to the fact that the gene, RL0189 which encodes gxrA (Fauvart et al., 2007).
was not induced in pea bacteroids (Karunakaran 2009).
Another interesting finding from the assay, is the high induction of enzyme activity using
Ketoglutaric acid using bacteroids compared to RLV 3841 which coincides with the
metabolomics data where it was detected to be 29 x more abundant compared to RLV 3841.
The possible reaction of this is ketoglutaric acid is converted to 2-Hydroxyglutarate by αketoglutarate dehydrogenase in the butanaote metabolism pathway (Figure 4).
As for the high induction of activity using glucarate in RLV 3841 and bacteroids, may not be a
genuine activity as the assay was only done once. The same goes for the high induction of
glycolic acid reductase in RLV 3841. These assay should be repeated again to obtained more
ascertain specific activity to prove the induction of any enzyme activity.
Figure 4: The butanoate metabolism pathway showing the reaction of α-ketoglutaratic acid (2oxoglutarate).
0.16
0.14
µmole/min/mg protein
0.12
0.1
0.08
RLV 3841 (Glucose)
RLV 3841 (Pyruvate)
0.06
0.04
RLV 3841 (Succinate)
Bacteroids
0.02
0
Figure 5: Enzyme specific rate activity when using different substrates of keto/hydroxy acids
with NAD+ as a co-substrate in free living Rhizobium leguminosarum bv. viciae strain 3841 and
Bacteroids
As for the assay using NAD+ as a co-substrate (Figure 5), there was high induction of the
activity for homoserine dehydrogenase (0.142 µmole/min/mg protein) and glyceraldehyde
dehydrogenase (0.112 µmole/min/mg protein) in bacteroids. However this assay would need to
be repeated to confirm the activity as it was only done once with one pool of bacteroids.
However from the metabolomics data, homoserine was detected to be 18.6x upfold in
bacteroids and 9500x in rhizosphere compared to RLV 3841 hence explaining the high
induction of activity. Looking at the highly upregulated genes in bacteroids from the
transcriptomic studies by Karunakaran et al., there is a gene which is 107 folds upregulated in
bacteroids and also upregulated in the rhizophere and only found in R.leguminosarum and R.etli
which encodes for a putative dehydrogenase. This gene is pRL100444 which might correspond
to the gene which encodes homoserine dehydrogenase. The suggested reaction for homoserine
is that it is converted to L-aspartate 4-semialdehyde by homoserine dehydrogenase in the
glycine, serine and threonine metabolism pathway (Figure 6).
Figure 6: The Glycine, serine and threonine metabolism pathway to show the reaction of
homoserine.
As for glyceraldehyde from the metabolomics data, it was detected by 2.8 x up in bacteroids
compared to RLV 3841, which might explain the high induction of glyceraldehyde acitivity. The
possible reaction for this activity is glyceraldehyde being converted to glyceric acid by glycerate
dehydrogenase (Figure 7)
Figure 7: The Glycolysis/Gluconeogensis metabolism showing the reaction of glyceraldehyde.
The results from the assay using NADP (Figure 8) and NADPH (Figure 9) as co-substrate show
a very low induction as the specific rate of activity was only between 0.001 – 0.008
µmole/min/mg protein. These assays need to be repeated to confirm whether the activity is a
genuine activity.
0.009
µmole/min/mg protein
0.008
0.007
0.006
0.005
0.004
RLV 3841 (Glucose)
0.003
RLV 3841 (Pyruvate)
0.002
RLV 3841 (Succinate)
0.001
Bacteroids
0
Figure 8: Enzyme specific rate activity when using different substrates of keto/hydroxy acids
with NADP as a co-substrate in free living Rhizobium leguminosarum bv. viciae strain 3841 and
Bacteroids.
µmole/min/mg protein
0.012
0.01
0.008
0.006
RLV 3841 (Glucose)
0.004
RLV 3841 (Pyruvate)
RLV 3841 (Succinate)
0.002
Bacteroids
0
Figure 9: Enzyme specific rate activity when using different substrates of keto/hydroxy acids
with NADPH as a co-substrate in free living Rhizobium leguminosarum bv. viciae strain 3841
and Bacteroids.
3.3 Citramalate assay
AA
0.25
B 0.0035
0.003
µmole/min/mg protein
µmole/min/mg protein
0.2
0.15
0.1
0.0025
0.002
0.0015
0.001
0.05
0.0005
0
0
3841
3841
3841
Bacteroids
(Glucose) (Pyruvate) (Succinate)
0.0025
D
0.002
µmole/min/mg protein
µmole/min/mg protein
C
3841
3841
3841 Bacteroids
(Glucose) (Pyruvate) (Succinate)
0.0015
0.001
0.0005
0.0025
0.002
0.0015
0.001
0.0005
0
0
3841
3841
3841 Bacteroids
(Glucose) (Pyruvate) (Succinate)
3841
3841
3841 Bacteroids
(Glucose) (Pyruvate) (Succinate)
Figure 10: Enzyme specific rate activity when using Citramalate as substrate in free living
Rhizobium leguminosarum bv. viciae strain 3841 and Bacteroid. (A) shows the specific rate
activity using NADH as co-substrate, (B) using NAD+ (C) using NADP and (D) using NADPH
labels gone wrong in D.
Citramalate activity was measured by adding citramalate to start the reaction and lactate
dehydrogenase was also added (draw a diagram to show the activity and show how the linking
enzymes work). There was a high induction of citramalate activity when using NADH as cosubstrate in both RLV 3841 and bacteroids. However, it was highly induced in bacteroids. There
are two possible reactions going on in this activity. One possibility of the reaction is citramalate
is converted to pyruvate and then to lactate as lactate dehydrogenase was added in the
reaction.
Other possibility of the reaction is condensation of citramalate to 2-methyl-maleate which is
involved in the making of (S)-3-Methyl-2-oxopentanoate, 2-Oxoisovalerate and 4-Methyl-2oxopentanoate which from the metabolomics data is only detected in bacteroids (Figure 11).
However, an interesting fact is that when the assay was done without adding lactate
dehydrogenase, there was no activity detected for this reaction and the same when citramalate
is no added. This shows that for this reaction to occur, it requires lactate dehydrogenase which
is an interesting fact further investigate.
Looking at the high induction of citramalate in both RLV 3841 and bacteroids using NADH as
co-substrate and at the transcriptomic data to link the activity. The gene PRL 120632 which
encodes for a putative dehydrogenase is good candidate to be the gene which encodes for the
induction of citramalate, as the gene was found highly upregulated in both Rhizobium and
bacteroids.
Figure 11: The Valine, Leucine and Isoleucine biosynthesis pathway to show the reaction of citramalate
(R-2-Methylmalate)
4. DISCUSSION
Transcriptomic and metabolomics studies on Rhizobium and bacteroids have provided
information on which genes were upregulated and metabolites which are formed during nitrogen
fixation. However, none of these studies could explain the metabolic processes of nitrogen
fixation particularly the enzyme activity. From the study, few interesting facts were discovered
which could lead to the linking of the transcriptomic data with the metabolomics data.
Several metabolites used in the study have showed high induction of activity which are
glyceraldehyde, homoserine and citramalate. Looking at the transcriptomic data, four genes
PRL100444, PRL120632, RL1694 and RL4267 was highly upregulated either in both Rhizobium
and bacteroids or in just bacteroids (Karunakaran et al., 2009). These genes could be potential
genes which encodes for the high induction of these metabolite activities.
As mention in the results part (3.2 and 3.3), the gene PRL100444 might encode for the
induction of homoserine and PRL120632 for the induction of citramalate. Further experiment
can be done to confirmed whether these genes does encodes for the induction of the
metabolites as the mutants of these genes are readily available from Phil poole lab. The mutant
bacteria could be inoculated onto the pea plant to see if there is any enzyme activity when using
its bacteroids or it can be done in the laboratory by inserting it into an expression plasmid to see
whether it is switched on.
Studies on amino acid transporter in bacteroids have been shown to be essential to transport
the branched chain amino acids (Prell et al., 2009). The information from this study might
support the fact that bacteroids requires amino acid transporter as it can be seen that bacteroids
accumulate the keto acids for these amino acids but seem unable to catalyze the final
transamination.
There are many potential ways of expanding the research in this study, it can be continued by
using other key metabolites which have been detected as high induced in bacteroids and
identify the potential reaction and genes which encodes it. Apart from that, amino acid
transferase activity could also be measured when doing the enzyme activity to see if the
enzyme is involved in any of the reactions.
If the study was to proceed, interesting key metabolites to look at are ononitol and pinitol which
are highly elevated in bacteroids. Ononitol was found the to be detected 62 x in bacteroids and
1000 x in the rhizosphere and to be found high in soy bean nodules. As for pinitol it was also
detected 1000x fold in bacteroids of soybean bacteroids but not pea bacteroids. This suggests
that peas only make ononitol but soybean make both compounds.
As there are many other key metabolites to studied and assayed, the development of a highthroughput method is necessary. This can be achieved by using a 96 cell well micro-titre plates.
The high throughput platform would be of great advantage to assay up to 50 different enzymes
at one time and reduce time taken to do the assay.