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.