KINETIC AND ENERGETIC ASPECTS

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

sufficient energy equivalents as this holds, e. g., to the variant with the 2-hydroxyisobutyrylCoA mutase have the potential to grow faster. This is supported by stoichiometric but also

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

dissimilate.

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.

CONCLUSIONS

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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779.

 

New Bacterial CoA-Carbonyl Mutases 85

Highly likely, 2-HIBA formation is not restricted to microbial MTBE degradation but

may also be an intermediate of other substances bearing a tert-butyl group as it is shown in

Figure 1. Besides MTBE, other ether compounds such as the fuel additive ethyl tert-butyl

ether (ETBE) are also degraded via TBA and 2-HIBA. In addition, it has been found that the

hydrocarbon compound isobutene can be transformed to 2-HIBA via isobutene epoxide and

2-hydroxy-2-methylpropanol (Henderson et al. 1993) (Figure 1). A third source for 2-HIBA

might be the degradation of the plant cyanoglycoside linamarin (Forslund et al. 2004). In the

course of its mineralization, 2-hydroxyisobutyronitrile is formed which could be transformed

into 2-HIBA by nitrilase activity (Banerjee et al. 2002) (Figure 1). However, there is no

evidence for a bacterium or other microorganism capable of growing on 2-

hydroxyisobutyronitrile and employing the mentioned nitrile-degrading enzyme.

C

CH3

CH2

H3C OH

HO

2-hydroxy-2-methylpropanol

H3C O CH2 R

CH3

CH3

H3C OH C

CH3

CH3

C

O

H3C

CH2

CH3

C

isobutene

epoxide

C

CH3

CH2

H3C

isobutene

alkyl tert-butyl

ether

tert-butyl

alcohol

3-hydroxybutyryl-CoA O

CoA-S

C

H

H2C OH

CH3

C

2-hydroxyisobutyryl-CoA

O

CoA-S

H3C OH

C

CH3

C

linamarin

2-hydroxyisobutyronitrile

C

CH3

CN

H3C OH

CoA-carbonyl mutase

Figure 1. Putative pathways of compounds containing a tert-butyl group or a related structure resulting

in the central intermediate 2-hydroxyisobutyryl-CoA which is proposed to be converted into 3-

hydroxybutyryl-CoA by CoA-carbonyl mutase activity.

In conclusion, the metabolism of several tert-butyl-containing compounds might result in

the formation of 2-HIBA as a central intermediate. By employing an ICM-like 2-

hydroxyisobutyryl-CoA mutase (Rohwerder et al. 2006), this recalcitrant carbonic acid can

be converted into the common metabolite 3-hydroxybutyrate, thus allowing complete

mineralization. Generally, main sources of 2-HIBA contamination in the environment can be

assumed to be industrial activities. Due to its widespread presence in groundwater systems,

MTBE may be the driving force for the evolution of this mutase pathway in the last decades.

However, a 2-hydroxyisobutyryl-CoA mutase might have evolved much earlier at sites where

wastewaters from methacrylate-producing plants had been treated.

86 Thore Rohwerder and Roland H. Müller

C

CH3

C

H3C CH

CoA-S

O

3

CoA-carbonyl mutase

C

CH3

H2C CH3

H

C

CoA-S

O

3-methylbutyryl-CoA

isooctane

C

CH3

CH2

H3C CH3

H3C CH3

CH3

C

H3C CH3

C5H11

CH3

C

H3C

CH3

CH

CH

COOH

H3C

H3C CH3

C

CH3

C

O

O

C

CH3

C

H3C CH3

COOH

CH2

pivalyl-CoA

2,2-dimethylheptane

C

CH3

H3C CH3

tert-butyl benzene

C O

H2C

CH2

H2C COOH

COH

Figure 2. Putative pathways of compounds containing a quaternary carbon atom resulting in the central

intermediate pivalyl-CoA which is proposed to be converted into 3-methylbutyryl-CoA by CoAcarbonyl mutase activity.

(b) Isomerization of Pivalic Acid

Pivalic acid (2,2-dimethylpropionic acid) is a highly branched short-chain carbonic acid,

which can be described as a quaternary carbon atom surrounded by three methyl moieties

plus the carboxyl group. Although it occurs in nature (Schiffman et al. 2001), a biosynthetic

pathway is not known. Therefore, it can be assumed that pivalate like 2-HIBA is mainly of

anthropogenic origin, e. g. pharmaceutical wastewater, as pivalic acid esters are established

prodrugs and produced in large quantities (Cherie Ligniere et al. 1987; Sauber et al. 1996;

Takada & Sudoh 2003). Since pivalate has been previously supposed to be completely

recalcitrant against microbial attack it is commonly used as a reference substance for

determining volatile fatty acid production in biological systems (Czerkawski 1976). Until

now, the bacterial degradation pathway has not been elucidated but only several routes have

been discussed (Probian et al. 2003; Solano-Serena et al. 2004). Obviously, the degree of

branching has to be reduced for further conversion. However, removing of a carbon atom

binding to the quaternary one, e. g. the carboxyl moiety by decarboxylation, requires

introduction of an additional functional group in the β-position. As hydroxylation reactions

are normally used for such an activating step, it can be suggested that pivalate persists

New Bacterial CoA-Carbonyl Mutases 87

especially in anoxic environments. However, anaerobic biodegradation has been reported in a

few cases (Chen et al. 1994; Perri 1997; Probian et al. 2003). Interestingly, in one proposal a

mutase reaction is thought to convert the CoA-activated pivalate to 3-methylbutyrate, very

similar to the recently identified 2-HIBA isomerization (Rohwerder et al. 2006) (Figure 1),

allowing further degradation even under anoxic conditions (Smith & Essenberg 2006) (Figure

2). Once more, this would be an example for the elegant employment of an 1,2-rearrangement

for transforming a highly branched into a less branched compound, i. e. in the case of

pivalate, from a quaternary into a tertiary carbon atom.

Besides its presence in pharmaceutical wastes, pivalate could be formed in the course of

bacterial conversion of branched hydrocarbon compounds containing quaternary carbon

atoms. Thus far, pivalate has been found to be an intermediate in aerobic isooctane (2,2,4-

trimethylpentane) degradation by Mycobacterium austroafricanum IFP 2173 (Solano-Serena

et al. 2004), and accumulation has been observed in aerobic cultures of Achromobacter

strains growing on 2,2-dimethylheptane or tert-butyl benzene (Catelani et al. 1977).

Unfortunately, further degradation of pivalate has not been elucidated in these studies and,

consequently, no pathway can be excluded at the moment. In agreement with the abovementioned assumption that additional groups are required in β-position for further

degradation, hydroxylation has been proposed for the aerobic pathway of pivalate (SolanoSerena et al. 2004). However, the finding of an employment of 2-hydroxyisobutyryl-CoA

mutase in the aerobic 2-HIBA pathway (Rohwerder et al. 2006) let us assume a similar

mutase activity for pivalate conversion. Hence, an isomerization reaction is likely not

restricted to anaerobic degradation but may also be responsible for the conversion under oxic

conditions (Figure 2).

(c) Isomerization of (1-Methylalkyl)- and (1-Phenylethyl)-Succinate

Contrary to the previous mutase reactions, this third example of novel cobalamindependent CoA-carbonyl mutases is obviously restricted to anoxic conditions as it is

employed in degradation pathways initiated by a special activation reaction, i. e. the addition

to fumarate, which is known to occur only in anaerobic bacteria. Besides other mechanisms,

an activating addition to fumarate was found in sulfate-reducing and denitrifying bacteria for

the conversion of alkanes (Callaghan et al. 2006; Cravo-Laureau et al. 2005; Wilkes et al.

2002) as well as ethylbenzene (Kniemeyer et al. 2003). In these cases, activation occurs at the

secondary carbon atom of the alkane chain and ethyl residue resulting in the formation of the

carbonic acids (1-methylalkyl)- and (1-phenylethyl)-succinate, respectively. Due to this

subterminal addition both intermediates contain two vicinal tertiary carbon atoms, thus

building up a structure which excludes conventional oxidation sequences. As mentioned

earlier, carbonic acids with a β-carbonyl function may easily undergo decarboxylation.

However, in (1-methylalkyl)- and (1-phenylethyl)-succinate the carbonyl group is not in the

β- but in the γ-position. Obviously, an 1,2-rearrangement catalyzed by CoA-carbonyl mutases

can solve the problem. Consequently, it has been proposed that after CoA-activation the

carboxyl group migrates and the less branched (2-alkylpropyl)- and (2-phenylpropyl)-

malonyl-CoA, respectively, are formed (eqs. 3 and 4). Then, these carbonic acids are

88 Thore Rohwerder and Roland H. Müller

decarboxylated and further degraded by β-oxidation. Although the responsible mutase

enzymes have not yet been identified, deuterium labeling experiments and metabolite

analysis unequivocally demonstrated the carbon skeleton rearrangement (Kniemeyer et al.

2003; Wilkes et al. 2002, 2003). In the case of ethylbenzene degradation via addition to

fumarate, (2-phenylpropyl)-malonyl-CoA is decarboxylated to 4-phenylpentanoyl-CoA

which, due to the phenyl group, can undergo only one β-oxidation step resulting in 2-

phenylpropionyl-CoA. Interestingly, it can, therefore, be speculated about a second CoAcarbonyl mutase reaction involved in this pathway for converting the 2-phenylpropionyl-CoA

to 3-phenylpropionyl-CoA, thus allowing a second round of β-oxidation (Kniemeyer et al.

2003). In addition to activation via addition to fumarate, other mechanisms exist for the

anaerobic degradation of alkanes, ethylbenzene and related compounds, e. g. activation via

carboxylation. At the moment, it is not clear whether these different mechanisms are

widespread and which pathway is most important (Callaghan et al. 2006). However, it is

likely that CoA-carbonyl mutase reactions play a significant role in anaerobic oxidation of

alkanes, which are major constituents of petroleum and natural gas.

(3)

O O

O S

CoA CoA

S

O

O

O

R

R

(1-methylalkyl)-succinyl-CoA (2-alkylpropyl)-malonyl-CoA

(4) O

O

O

S

CoA CoA

O S

O O

(1-phenylethyl)-succinyl-CoA (2-phenylpropyl)-malonyl-CoA

STRUCTURAL AND EVOLUTIONARY ASPECTS

Prokaryotic MCMs are organized as homo- or heterodimers with a subunit size of about

700 amino acids (Birch et al. 1993; Trevor & Punita 1999) (Figure 3). The substrate- and

cobalamin-binding domain sequences are highly conserved. In case of heteromeric structure,

only one polypeptide strand contains the functional domains, e. g. the MCM large subunit in

P. shermanii (Marsh & Leadlay 1989; Marsh et al. 1989), whereas the other subunit does not

bind methylmalonyl-CoA and cobalamin but is thought to generally stabilize the enzyme

complex. In contrast to MCM, thus far identified ICM structures are heterodimers where

New Bacterial CoA-Carbonyl Mutases 89

substrate- and cobalamin-binding domains are located on the different polypeptide strands,

respectively. Consequently, the substrate-binding large subunit (IcmA, about 570 amino

acids) is very similar to the N-terminal segment of the MCM polypeptide whereas the

cobalamin-binding small subunit (IcmB, about 140 amino acids) is nearly identical to the Cterminal domain of MCM (Ratnatilleke et al. 1999; Zerbe-Burkhardt et al. 1998) (Figure 3).

Y89 B12 domain

IcmB

R207

F80 Q198

IcmA

B12 domain

MCM large subunit

Figure 3. Comparison of the structural organization of MCM (MCM large subunit of P. shermanii) and

ICM (IcmA and IcmB from S. cinnamonensis). Amino acid residues important for substrate binding are

indicated.

Substrate binding and reaction mechanism in CoA-carbonyl mutases has only been

studied in detail for MCM (Gruber & Kratky 2001; Mancia et al. 1999). In brief, the

homolysis of the cobalt-carbon bond produces an adenosyl radical that abstracts a hydrogen

atom from the substrate, resulting in a substrate-derived radical intermediate. After

rearrangement, a product-related radical retrieves the hydrogen atom from the adenosyl group

and the product is formed. The resulting adenosyl radical can again combine with the cobalt

cofactor, bringing the reactive site back into the initial state. Due to the radical nature of the

rearrangement mechanism protection of the highly reactive intermediates is required and,

consequently, the reaction takes place deeply buried within the enzyme. In contrast, the initial

structure of the reactive site has to be easily accessible for substrates. Hence, a significant

conformational change can be observed after substrate has bound to the enzyme, in the course

of which the adenosyl radical is formed and the reaction gets started. The mayor catalytic

function of the enzyme may be just holding the substrate and product-related radicals in the

correct orientation (Mancia et al. 1999). For doing this, certain amino acids specifically

interact with substrate and intermediates. In particular, the highly conserved Arg207 and

Tyr89 of MCM (MCM large subunit, P. shermanii numbering) hold the free carboxyl group

of methylmalonyl-CoA while Gln197 binds to the thioester group. According to the differing

substrate isobutyryl-CoA, lacking a free carboxyl group, in the ICM sequences thus far

identified, Arg207 and Tyr89 of MCM are replaced by Gln198 and Phe80 (ICM large subunit

IcmA, S. cinnamonensis numbering) (Ratnatilleke et al. 1999; Zerbe-Burkhardt et al. 1998),

respectively. Obviously, the positively charged guanidino group of Arg and the polar phenol

moiety of Tyr are not required for holding the methyl residues of isobutyryl-CoA or would

even prevent binding of these nonpolar groups (Mancia et al. 1999; Ratnatilleke et al. 1999).

90 Thore Rohwerder and Roland H. Müller

Besides holding the substrate, Tyr89 of MCM and the corresponding Phe80 of ICM are

thought to be also important for stereochemistry of the rearrangement reaction and for driving

off the adenosyl group from the cobalt cofactor (Mancia et al. 1999). However, other

substrates may require further modifications at this important reactive site position. In the

newly discovered ICM-like mutase converting 2-hydroxyisobutyryl-CoA, a corresponding

Ile90 (ICM large subunit IcmA, M. petroleiphilum PM1 numbering) has been found

(Rohwerder et al. 2006) (Figure 4). Possibly, the quite large phenyl moiety of Phe80 of ICM

does not allow the binding of the 2-hydroxyisobutyryl residue in contrast to isobutyryl-CoA,

lacking the hydroxyl group. Interestingly, database research on CoA-carbonyl mutase-like

sequences reveals only less than a handful enzymes where the Tyr89 of MCM or Phe80 of

ICM is replaced with Ile (Figure 4). It remains to be elucidated whether this replacement

results in a different substrate specificity allowing the conversion of 2-hydroxyisobutyrylCoA.

Figure 4. CLUSTAL W alignment of the reactive site segment of MCM large subunit from P.

shermanii (X14965), ICM large subunit from S. cinnamonensis (AAC08713) and ICM-like large

subunit of 2-hydroxyisobutyryl-CoA mutase from M. petroleiphilum PM1 (ZP_00242470). The Tyr89

of MCM and the corresponding Phe80 of ICM and Ile90 of 2-hydroxyisobutyryl-CoA mutase are in

boldface. The only BLAST matches of ICM-like sequences showing the Ile90 are from Rhodobacter

sphaeroides ATCC 17029 (EAP67072), Xanthobacter autotrophicus Py2 (EAS17594) and

Nocardioides sp. JS614 (EAO08692).

Unfortunately, the structure of other CoA-carbonyl mutases, such as the above proposed

pivalyl-CoA, (1-methylalkyl)-succinyl-CoA and (1-phenylethyl)-succinyl-CoA mutases, has

not been characterized thus far. However, on the basis of the known features of the reaction

centers of MCM, ICM and 2-hydroxyisobutyryl-CoA mutase one might also speculate about

the structure of the other enzymes. Hence, the substrate-binding site of pivalyl-CoA mutase

should resemble the one of ICM due to the high structural similarities of their substrates,

pivalyl-CoA and isobutyryl-CoA, respectively. Accordingly, equivalents of Gln198 and

Phe80 of ICM are expected to be present for interacting with the three methyl moieties of

pivalyl-CoA. In (1-methylalkyl)-succinyl-CoA and (1-phenylethyl)-succinyl-CoA mutases,

on the other hand, equivalents of Arg207 and Tyr89 of MCM are supposed to be found as

their substrates have the same dicarboxylic acid structure. Besides these proposed similarities

with MCM, significant sequence deviations are expected, of course, due to the quite large

side chain of their substrates, the (1-methylalkyl) and (1-phenylethyl) residues, compared

with the corresponding hydrogen atom in succinyl-CoA. Generally, it can be assumed that

most of the here proposed novel CoA-carbonyl mutases, such as 2-hydroxyisobutyryl-CoA

P. shermanii MCM: ATMYAFRPWTIRQYAGFSTAKESNAFYRRN

S. cinnamonensis ICM: ATGYRGRTWTIRQFAGFGNAEQTNERYKMI

M. petroleiphilum ICM-like: PTMYRSRTWTMRQIAGFGTGEDTNKRFKYL

R. sphaeroides ICM-like: PTMYRGRNWTMRQIAGFGTGEDTNKRFKFL

X. autotrophicus ICM-like: PTMYRSRNWTMRQIAGFGTGEDTNKRFKYL

Nocardioides sp. ICM-like: PTMYRGRHWTMRQIAGFGQAEETNKRFQYL

New Bacterial CoA-Carbonyl Mutases 91

mutase, are not structurally related to MCM but to ICM. Then, these enzymes would be made

up of a substrate-binding large subunit (IcmA-like) and a cobalamin-binding small one

(IcmB-like) which would be encoded by two distinct structural genes. This organization

enables an independent replication of these two functions. Hence, in comparison with MCM

a ICM-like genetic structure allows more flexibility for the evolution of a new substrate

specificity.

 

ICM

isobutyryl-CoA butyryl-CoA

H

H3C COSCoA

CH3

H

H3C

COSCoA

H2C

H

First evidence of this mutase reaction was provided by Robinson and coworkers (Gani et

al. 1985; Reynolds et al. 1988), while studying the synthesis of the antibiotic monensin A in

Streptomyces cinnamonensis A3823.5. The carbon backbone of this polyether antibiotic is

assembled from 5 acetate, 1 butyrate and 7 propionate building blocks, indicating the

importance of propionate metabolism. In the course of valine degradation, isobutyrate is

formed and linked to butyrate metabolism by ICM activity. Then, propionate may be

produced either directly by α-oxidation or succinate is formed by ω-oxidation (Reynolds et

al. 1988). The latter can be transformed to propionate via methylmalonyl-CoA. Hence,

monensin A biosynthesis involves both MCM and ICM activities.

In addition to antibiotic production in Streptomyces spp., ICM activity was found to be

involved in the primary metabolism of several anaerobic bacterial strains and enrichment

cultures. As isobutyrate cannot be directly degraded via β-oxidation isomerization to butyrate

seems to be a possible pathway in anoxic environments, enabling growth on isobutyrate

sources such as valine. Consequently, this conversion has been detected under sulfatereducing as well as methanogenic conditions (Angelidaki & Ahring 1995; Oude Elferink et

al. 1996; Tholozan et al. 1988; Wu et al. 1994). Although ICM has not been biochemically

and genetically characterized in these anaerobic cultures, the isomerization reaction was

unambiguously proved by 13C labeling experiments, e. g., in Desulforhabdus amnigena DSM

10338 (Oude Elferink et al. 1996) and in a methanogenic enrichment (Tholozan et al. 1988).

In the latter case, syntrophic bacteria living in cooperation with methanogenic species may be

responsible for isobutyrate isomerization, such as the strict anaerobic glutarate-fermenting

bacterium Pelospora glutarica WoG13 (Matthies & Schink 1992; Matthies et al. 2000) and

the thermophilic, fatty acid-oxidizing Synthrophothermus lipocalidus TGB-C1 (Sekiguchi et

al. 2000). Thus, ICM may play a significant role for isobutyrate turnover in biotopes where it

is formed under anoxic conditions, e. g., in the course of anaerobic valine degradation.

Both mutase enzymes, MCM and ICM, have a rather limited substrate spectrum and

function effectively merely with their natural substrates methylmalonyl-CoA and isobutyrylCoA, respectively (Mancia et al. 1999). At least by MCM, a few other substrates are known

to be transformed albeit at far lower turnover rates than methylmalonyl-CoA, e. g., glutarylCoA, methylsuccinyl-CoA and ethylmalonyl-CoA (Padmakumar & Banerjee 1995; Rétey et

al. 1978; Shinichi et al. 1994). However, considering the elegant way by which

methylmalonate and isobutyrate are converted into their corresponding straight-chain

84 Thore Rohwerder and Roland H. Müller

carbonic acids, one might speculate about similar 1,2-rearrangements to be involved in other

pathways. Indeed, this kind of reaction would be suitable for degrading hydrocarbons with

tertiary or even quaternary carbon atoms, where a further oxidation is prevented and deadend products are reached, e. g., due to an impossible β-oxidation at the branched position.

However, until recently other CoA-carbonyl mutases than MCM and ICM have not been

identified and often mutase reactions were not considered when bacterial degradation

pathways were investigated. This conclusion might turn out to be wrong as new findings let

us claim that ICM-like enzymes could perfectly well function in many degradation pathways.

For substantiating this hypothesis, we will present in the following three examples for

possible employment of thus far unknown bacterial CoA-carbonyl mutases; the conversion of

(a) 2-hydroxyisobutyrate, (b) pivalate and (c) carbonic acid intermediates of anaerobic alkane

and ethylbenzene degradation.

NOVEL COA-CARBONYL MUTASES

(a) Isomerization of 2-Hydroxyisobutyrate

The tertiary carbon atom-containing 2-hydroxyisobutyrate (2-HIBA) is rarely found in

nature and only few applications of this branched carbonic acid are known, such as the use of

its complexing properties for analyzing rare earth elements (Raut et al. 2002). In addition, it

is an intermediate and by-product of industrial processes, such as the synthesis of

methacrylate in the classical acetone cyanohydrin process since the mid-1930s (Chisholm

2000). As 2-HIBA seems not to be a widespread contaminant the investigation of its

degradation by bacteria or other microorganisms has not attracted much attention. This

situation changed in the last years since 2-HIBA has been identified as an intermediate in the

degradation pathway of the fuel oxygenate methyl tert-butyl ether (MTBE) (Fayolle et al.

2001). Due to its massive use since the 1990s, the current world production amounts to about

20 Mt/a, MTBE has become a common groundwater contaminant and thus severely threatens

drinking water resources by its suspected carcinogenicity, as well as by its unpleasant odor

and taste (EPA 1997; Moran et al. 2005; Schmidt et al. 2002). Consequently, a main concern

is the environmental fate of the fuel oxygenate and its degradation intermediates. As in situ

biodegradation is the only sustainable sink of MTBE in aquifers extensive research work is

currently undertaken for elucidating its microbial degradation pathway. Aerobically, MTBE

is degraded via tert-butyl alcohol (TBA) and 2-hydroxy-2-methylpropanol to 2-HIBA

(Fayolle et al. 2001; Lopes Ferreira et al. 2006; Steffan et al. 1997) (Figure 1). In the first

investigations on bacterial MTBE degradation, further steps were not identified but only

three possible routes were proposed starting with hydroxylation, dehydration or

decarboxylation of 2-HIBA (Steffan et al. 1997). However, these mechanisms have not been

proved until now. On the contrary, evidence for a fourth pathway involving the activity of a

CoA-carbonyl mutase has been furnished (Rohwerder et al. 2006). The novel ICM-like

mutase catalyzes the conversion of 2-HIBA into 3-hydroxybutyrate (Figure 1) and, thus,

connects the MTBE-specific degradation steps with the common metabolism.

 

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Chapter V

NEW BACTERIAL COBALAMIN-DEPENDENT

COA-CARBONYL MUTASES INVOLVED IN

DEGRADATION PATHWAYS

Thore Rohwerder1,∗ and Roland H. Müller2

1

Aquatic Biotechnology, Biofilm Centre, University Duisburg-Essen, Geibelstr. 41, D47057 Duisburg, Germany;

2

Department of Environmental Microbiology, Helmholtz Centre for Environmental

Research UFZ, Permoserstr. 15, D-04318 Leipzig, Germany.

ABSTRACT

The adenosylcobalamin-dependent CoA-carbonyl mutases catalyze the 1,2-

rearrangement of carbonyl groups reversibly converting branched-chain carbonic acids

into straight-chain ones. Currently, this enzyme group comprises of only two known

mutases, the extensively studied methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) and

isobutyryl-CoA mutase (ICM, EC 5.4.99.13). Whereas MCM is widespread among

bacteria and animals ICM seems to be restricted to bacteria and has thus far only been

characterized in Streptomyces spp. Both enzymes have a rather limited substrate

spectrum and function effectively merely with their natural substrates methylmalonylCoA and isobutyryl-CoA, respectively. Interestingly, we have recently discovered a

novel bacterial CoA-carbonyl mutase catalyzing the conversion of 2-hydroxyisobutyrylCoA into 3-hydroxybutyryl-CoA (Rohwerder et al. 2006, Appl. Environ. Microbiol.

72:4128). This enzyme plays a central role in the productive degradation of compounds

containing a tert-butyl group such as the common fuel additives methyl and ethyl tertbutyl ether. Similar enzymes are proposed to be involved in the conversion of pivalic

acid and in the degradation of alkanes and alkylated aromatic hydrocarbons via anaerobic

pathways employing addition to fumarate. Since all these compounds are important

∗

 Correspondence concerning this article should be addressed to: Thore Rohwerder, Aquatic Biotechnology,

Biofilm Centre, University Duisburg-Essen, Geibelstr. 41, D-47057 Duisburg, Germany, e-mail:

thore.rohwerder@uni-due.de.

82 Thore Rohwerder and Roland H. Müller

pollutants of water and soil, cobalamin and the new CoA-carbonyl mutases play a thus

far not realized role in natural as well as induced bioremediation processes. Therefore,

we summarize in this chapter the known reactions and also speculate about further

pathways which have not yet been associated with CoA-carbonyl mutase activity. In

addition, the enzyme structure and the herewith possibly associated evolution of substrate

specificity are outlined. Finally, energetic and kinetic consequences are discussed which

may result from employing a cobalamin-dependent enzyme for dissimilatory pathways.

INTRODUCTION

The cobalamin-dependent CoA-carbonyl mutases are a group of enzymes catalyzing the

spectacular 1,2-rearrangement of the CoA thioester moiety of CoA-activated carbonic acids.

In this reaction, the cofactor adenosylcobalamin is used to create radical intermediates.

Currently, only two representatives of this enzyme group are known, the extensively studied

and widespread methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) and isobutyryl-CoA

mutase (ICM, EC 5.4.99.13). MCM catalyzes the reversible and stereospecific conversion of

(R)-methylmalonyl-CoA into succinyl-CoA (eq. 1).

H

-

OOC COSCoA

CH3

H

-

OOC

COSCoA

H2C

H

MCM

(R)-methylmalonyl-CoA succinyl-CoA

(1)

This mutase has been found in a variety of bacteria and also in animals, in which the

enzyme is located in the mitochondria (Gruber & Kratky 2001). The animal MCM is

involved in the conversion of branched-chain amino acids, odd-chain fatty acids and

cholesterol via propionate and methylmalonyl-CoA into succinyl-CoA (Willard & Rosenberg

1980). Any impairment of the mutase results in a serious disorder of organic acid metabolism

termed methylmalonic aciduria (Deodato et al. 2006). In contrast, bacterial propionate

degradation is much more diverse and, consequently, MCM catalysis is not always required

(Textor et al. 1997). However, additional functions of bacterial MCMs are known, including

also the catalysis of the reverse reaction from succinyl-CoA to methylmalonyl-CoA. For

example, in some fermenting bacteria, such as Propionibacterium shermanii, propionate is

not a metabolic intermediate but the end product of the anaerobic degradation of lactate or

pyruvate. Here, MCM is employed for synthesizing propionate from tricarboxylic acid-cycle

(TCC) intermediates via methylmalonyl-CoA (Allen et al. 1964). In Streptomyces spp., MCM

together with ICM is involved in secondary metabolism, providing building blocks for

polyketide antibiotic synthesis (Birch et al. 1993).

In contrast to MCM, ICM seems to be restricted to bacteria and has thus far only been

characterized in the above mentioned Streptomyces spp. (Ratnatilleke et al. 1999; Zerbe-

New Bacterial CoA-Carbonyl Mutases 83

Burkhardt et al. 1998). It catalyzes the reversible conversion of isobutyryl-CoA into butyrylCoA. Although stereospecificity is not as stringent as in MCM, an analogous retention of

configuration is also predominating in ICM (Moore et al. 1995) (eq. 2).

(2)