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.