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.
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
(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,
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
(c) Isomerization of (1-Methylalkyl)- and (1-Phenylethyl)-Succinate
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-
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.
(1-methylalkyl)-succinyl-CoA (2-alkylpropyl)-malonyl-CoA
(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
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
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
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