Besides the above mentioned structural and evolutionary aspects, growth of
microorganisms on pollutants which includes a cobalamin-dependent mutase step in their
primary degradative pathway was evaluated from kinetic and energetic viewpoints in this
chapter. At first glance, employing an adenosylcobalamin-dependent step not for secondary
metabolism but for the primary one resulted in an negative effect on bacterial growth in case
de novo synthesis of the cosubstrate is required. Mutase pathways, on the other hand, can be
more efficient for growth when compared with alternative routes, such as hydroxylation and
decarboxylation steps, as will be outlined in the following. Due to the rare data on other
degradation pathways special features were discussed mainly based on the example of MTBE
and 2-HIBA degradation (see mutase example a).
Growth rates on compounds with tertiary carbon structure were found in general to be
low; whenever growth was observed the rates amounted to 0.01 h-1 to 0.06 h-1. The general
deficit in microbial MTBE utilization was supported by the fact that degradation was possible
by a variety of strains and consortia only in the presence of a growth supporting substrate.
This indicates that the flows of carbon and energy resulting from MTBE degradation were
too low in the latter cases to support growth. The appearance of metabolites during the
degradation of MTBE hints moreover to imbalances in the substrate conversion and defines
bottlenecks in metabolism. One of such metabolites which were occasionally found in the
culture medium is 2-HIBA (François et al. 2002; Rohwerder et al. 2006; Steffan et al. 1997)
attributing the metabolic deficit to enzymatic step(s) involved in the conversion of this
intermediate. With the MTBE-degrader Aquincola tertiaricarbonis L108 (Lechner et al.
2007) the growth rates on MTBE and TBA amounted to 0.06 h-1 and 0.07 h-1, respectively, in
mineral salts medium supplemented with cobalamin. This vitamin was essential for growth.
Substitution by Co2+ seems in general to be possible but the degradation of MTBE was
difficult to stabilize under these conditions. The dependency of growth on these supplements
was correlated to the role of a special mutase in the MTBE metabolism which was detected in
certain MTBE-degrading strains and connects 2-HIBA as a central intermediate to the
general metabolism (Rohwerder et al. 2006) (Figure 1). Applying 2-HIBA as growth
substrate, which is after CoA activation the actual substrate of the 2-hydroxyisobutyryl-CoA
mutase, resulted in a maximum growth rate µmax in the presence of cyanocobalamin of 0.14 h1
, whereas this rate was reduced to about 0.055 h-1 when the vitamin B12 was substituted by
Co2+ (Rohwerder et al. 2006). Although it is in general difficult to attribute limits in the
growth rate to a defined step or sequence, the present results suggest that the availability of
cobalamin should exert such a role. Consequently, the coupling of the primary assimilatory
92 Thore Rohwerder and Roland H. Müller
route to parts in the secondary metabolism with special function, i. e. to those in the present
case which were involved in the synthesis of cobalamin, might control the overall substrate
conversion. This seems plausible and is in accordance with the position of this mutase
reaction as catalyzing a key step in a primary degradative pathway. The effect on growth rate
seems strongly to be correlated to the heterotrophic metabolism as outlined.
In general, growth of microorganisms on heterotrophic substrates is a trade-off between
rate and yield (Pfeiffer & Bonhoeffer 2002). This results from the fact that the metabolism of
heterotrophic substrates must deliver and equilibrate both carbon and energy for biomass
synthesis and maintenance. Accordingly, the metabolic branches for energy generation and
biosynthetic purposes are interconnected more or less tightly. Heterotrophic growth seems in
general to be energy-limited. This results from the fact that the energy content of substrates is
in most cases low or made available to only a limited extent during metabolism and through
coupling of energy transduction by oxidative phosphorylation. Thus the growth rate on a
heterotrophic substrate seems to be directly correlated to the energy production rate. In this
context, MTBE and other oxygenates are of exception when considered as heterotrophic
substrates. Stoichiometric calculations revealed that the carbon and energy was almost
balanced during degradation via defined pathways. This holds for instance to a pathway with
the 2-hydroxyisobutyryl-CoA mutase as the key step (Müller et al. 2007). This means that
energy equivalents were generated during assimilation of this compound which were
sufficient to incorporate carbon into biomass. This should have consequences with respect to
maximizing substrate conversion and its coupled energy production rates reasoned by several
facts. Assimilatory efforts in heterotrophic metabolism must in each case satisfy the
requirement of carbon precursors for biomass synthesis. If this process is at the same time
coupled to the energetic efforts and results in energy to an amount as required for biomass
synthesis, there will be no need to dissimilate additional substrate to CO2 merely for energy
generation. This should speed up the degradation rate for several reasons. In general,
common sequences are used for anabolism and catabolism to convert a substrate into
common metabolites. This holds above all with xenobiotic compounds where so-called
peripheral or upper pathways are usually applied to channel potential substrates into the
general metabolism. The capacities of the peripheral pathways may be considered as limiting,
as these routes are likely to be initially based on the fortuitous use of enzymes (Janssen et al.
2005). Flow of substrates by using these sequences comply assimilatory and dissimilatory
purposes. Exhaustion of the metabolic capacity consequently means that the supply of carbon
for assimilation is reduced when substrate is needed for dissimilation and hence growth rate
will be reduced. Accordingly, bacterial strains using an assimilatory sequence that delivers
kinetic terms (Müller et al. 2007).
Kinetic theory states that flux rates through a sequence are the lower the higher the
number of steps that are involved to convert a substrate to a product (Costa et al. 2006). This
means in the context of growth on MTBE by using the mutase pathway, that the formation of
energy equivalents through oxidizing substrate via e. g. TCC are not essentially required.
Consequently, the primary metabolic pathway is shortened and almost restricted to the
assimilatory route. This should result in a positive effect on the growth rate. In contrast,
New Bacterial CoA-Carbonyl Mutases 93
MTBE degradation via alternative routes (Steffan et al. 1997) with e. g. a decarboxylase or a
monooxygenase reaction as the key step for channeling 2-HIBA into the general pathway is
energetically less efficient (Müller et al. 2007). Consequently, additional substrate is needed
for dissimilation via the TCC in order to meet the overall energy requirement. When we take
into account the exhaustion of the upper pathway during conversion of MTBE up to 2-HIBA
as discussed above, both effects add up and should lead to a reduction of the overall growth
rate, the extent of which being increased the higher the portion of substrate is needed to
The same arguments with respect to pathway length and rate effects should apply to the
reduction of the rate observed by omitting cobalamin. Addition of Co2+ was a minimum
requirement to enable growth of strain L108 on substrates with tertiary carbon atom structure
(Rohwerder et al. 2006), whereas this trace element was not required during growth on
acetate (Müller et al. 2007). The fact that the growth rate was increased by adding cobalamin
was directly correlated to the function of the 2-hydroxyisobutyryl-CoA mutase and indicates
a strong coupling of the overall metabolism to the synthesis of the cosubstrate
adenosylcobalamin. The corresponding pathway is rather complex and includes a set of up to
20 to 30 enzymes (Martens et al. 2002). It is likely that the level of these enzymes and the
pools of very specific metabolites required for the synthesis of adenosylcobalamin will be
small and equilibrated to the overall equipment of the cell. This results from the fact that the
protein concentration of a cell is almost constant and sequences can be adjusted to the
requirement only in proportion to other enzymes. The actual concentration of the enzymes for
cobalamin synthesis is not known. It cannot be excluded, however, that the concentration of
cobalamin or its synthesis rate, respectively, were stimulated in a feed forward control pattern
in correlation to the need in the primary pathway. Thus, the observed overall growth rate
might even be due to elevated activity with respect to cobalamin supply in comparison to the
level of the enzymes for cobalamin synthesis under growth conditions in which mutase
reactions played only a secondary role.
The degradation pathways of MTBE are not yet completely resolved. So, it is not known
whether the pathway including the mutase step is the only sequence by which 2-HIBA can be
converted in a defined bacterial strain, as for instance A. tertiaricarbonis L108, or whether
there exist alternative routes that may function under defined conditions or in parallel. As
MTBE was only recently released into the environment, autarkic growth on this compound
seems to be the result of current evolution which might concern various enzymes in the
peripheral pathway. It is in general stated that xenobiotic compounds are degraded by the
fortuitous use of pre-existing enzymes (Janssen et al. 2005) and that selective advantage
comes into play to the extent that growth rate is increased. This includes mutation of the
enzymes which seems to be the case with the 2-hydroxyisobutyryl-CoA mutase. It became
evident that essential amino acids which should define substrate specificity were exchanged
in the ICM-like mutase from MTBE-degrading strains such as A. tertiaricarbonis L108 and
M. petroleiphilum PM1 in comparison to those of the known ICM (Rohwerder et al. 2006)
(Figure 4). Autarkic growth presumes that the net rates of energy production compensate at
least for the maintenance requirements. Otherwise growth would be negative as was shown in
a calculation with MTBE in which the thresholds for growth were defined by taking into
account relevant kinetic constants and the amounts of energy gained through use of various
94 Thore Rohwerder and Roland H. Müller
putative degradation routes (Müller et al. 2007). These results made evident that strains
which applied a pathway with 2-hydroxisobutyryl-CoA mutase as a key step (Figure 1)
showed advantages in the competition compared to some other putative routes.
The 1,2-rearrangement catalyzed by adenosylcobalamin-dependent CoA-carbonyl
mutases is not restricted to the conversion of isobutyrate and methylmalonate. On the
contrary, it is suggested in this chapter that the mutase activity is rather widespread among
bacteria and is employed for degrading highly branched organic compounds. Thus far, only
one of these novel enzymes has been identified catalyzing the conversion of 2-
hydroxyisobutyryl-CoA to 3-hydroxybutyryl-CoA. Often, the employment of an 1,2-
rearrangement reaction replaces alternative activation steps such as hydroxylation and
decarboxylation. Consequently, the mutase step is especially suitable for anoxic conditions
where activation by oxygen is not applicable. However, the 1,2-rearrangement has been
demonstrated also in an aerobic pathway where the fuel oxygenate MTBE is degraded via 2-
hydroxyisobutyryl-CoA. It is proposed that the novel CoA-carbonyl mutases have a ICM-like
structure consisting of a substrate-binding large subunit and a cobalamin-binding small
subunit. In contrast to MCM, this structural organization allows more flexibility for the
evolution of a new substrate specificity. On principle, employing a cobalamin-containing
enzyme for primary metabolism is a burden as de novo synthesis of the cosubstrate is quite
costly for the bacterial cell due to the large number of enzymatic steps which are involved.
Nevertheless, the use of a CoA-carbonyl mutase can obviously prevail over energetically less
efficient alternative routes employing hydroxylation or other activation mechanisms, as has
been demonstrated for the example of MTBE and 2-HIBA degradation. Hence, cobalamin
and CoA-carbonyl mutase may play an important role in the turnover of branched compounds
of natural origin as well as anthropogenic sources.
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