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W. (1997). Propionate oxidation in Escherichia coli: evidence for operation of a

methylcitrate cycle in bacteria. Arch. Microbiol., 168, 428–436.

Tholozan, J.-L.; Samain, E.; Grivet, J.-P. (1988). Isomerization between n-butyrate and

isobutyrate in enrichment cultures. FEMS Micobiol. Lett., 53, 187-191.

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In: Vitamin B: New Research ISBN 978-1-60021-782-1

Editor: Charlyn M. Elliot, pp. 99-119 © 2008 Nova Science Publishers, Inc.

Chapter VI

CYSTALYSIN: AN EXAMPLE OF THE CATALYTIC

VERSATILITY OF PYRIDOXAL 5’-PHOSPHATE

DEPENDENT ENZYMES

Barbara Cellini∗ , Riccardo Montioli and Carla Borri Voltattorni

Dipartimento di Scienze Morfologico-Biomediche, Sezione di Chimica Biologica,

Facoltà di Medicina e Chirurgia, Università degli Studi di Verona, Strada Le Grazie, 8,

37134 Verona, Italy.

ABSTRACT

Pyridoxal 5’-phosphate (PLP) is the catalitically active form of the water-soluble

vitamin B6, and hence the cofactor of a number of enzymes essential to the human body.

PLP-dependent enzymes are unique for the variety of reactions on amino acids that they

are able to catalyze (transamination, decarboxylation, racemization, β- or γreplacement/elimination). In the absence of the apoenzyme, different reactions would

occur simultaneously, but the protein moiety drives the catalytic power of the coenzyme

toward a specific reaction. However, this specificity is not absolute; most PLP-enzymes

catalyze indeed side-reactions which can have physiological significance and provide

interesting mechanistic and stereochemical information about the structure of the enzyme

active site.

Cystalysin is a PLP-dependent Cβ-Sγ lyase present in Treponema denticola, and its

main reaction is the α,β-elimination of L-cysteine to produce pyruvate, ammonia and

H2S. The latter is probably responsible for the hemolytic and hemoxidative activity

associated with the enzyme catalysis. Cystalysin is one of the most representative

examples of the high catalytic versatility of PLP-dependent enzymes. Recently, indeed, it

has been shown that cystalysin is also able to catalyze the racemization of both

∗

 Correspondence concerning this article should be addressed to: Barbara Cellini, Dipartimento di Scienze

Morfologico-Biomediche, Sezione di Chimica Biologica, Facoltà di Medicina e Chirurgia, Università degli Studi

di Verona, Strada Le Grazie, 8, 37134 Verona, Italy. Tel.: +39-045-8027-293; Fax: +39-045-8027-170; E-mail:

barbara.cellini@univr.it.

100 Barbara Cellini, Riccardo Montioli and Carla Borri Voltattorni

enantiomers of alanine, the β-desulfination of L-cysteine sulfinic acid, and the βdecarboxylation of L-aspartate and oxalacetate with turnover numbers measured in

seconds, and the transamination of L- and D-alanine with turnover numbers measured in

minutes.

Extensive biochemical investigations have uncovered several interesting features of

cystalysin, including the binding mode of the cofactor, its substrate specificity, the

formation of reaction intermediates characteristic of most PLP-enzymes, and the

involvement of some active-site residues in the primary and secondary catalytic

reactions.

INTRODUCTION

Vitamin B6 is a water-soluble compound discovered about 70 years ago whose major

active chemical form is pyridoxal 5’-phosphate (PLP), that plays a vital role as a cofactor of a

large number of enzymes in all organisms [1]. Overall, the Enzyme Commission (EC;

http://www.chem.qmul.ac.uk/iubmb/enzyme/) has listed more than 140 PLP-dependent

enzymatic activities, corresponding to about 4% of all classified activities. Additionally,

several putative PLP-binding proteins have been identified in genome sequencing projects

[2]. PLP is considered to be one of the nature’s most versatile cofactors, and PLP-dependent

enzymes mediate different cellular processes mainly involving amino compounds and

ranging from the biosynthesis of amino acids and amino acids-derived metabolites, to the

biosynthesis of amino sugars and other amino-containing compounds [3]. They catalyze a

wide variety of reactions, including transamination, racemization, decarboxylation, β- or γreplacement/elimination, and provide a unique model to understand the mechanisms by

which enzymes control substrate and reaction specificity [4]. In all PLP-enzymes, the

cofactor is covalently bound to the apoprotein through a Schiff base linkage between the

aldehydic group of the coenzyme and the ε-amino group of an active site lysine residue

(internal aldimine). With the exception of phosphorilases, which utilize PLP in a different

way and will not be considered here, the first step is common to all PLP-catalyzed reactions

and consists in the displacement of the active site lysine by an incoming substrate amino

group to form the external aldimine [1]. From this point on, the catalytic pathways differ

among the enzymes according to their reaction specificity. In fact, in the next step of the

reaction, each one of the three bonds at Cα of the external aldimine may be broken resulting

in the formation of a quinonoid intermediate. This process is facilitated by the electron-sink

properties of the pyridine moiety of the coenzyme, which stabilizes the developing negative

charge. On the basis of the Dunathan’s hypothesis [5], advanced in 1966 and later confirmed

by the resolution of the aspartate aminotransferase/phosphopyridoxyl aspartate complex [6],

the bond to be cleaved is the one aligned perpendicularly to the pyridine ring of the cofactor.

This allows the resulting carbanion to be stabilized by conjugation with the extended πsystem of PLP. The topology of the external aldimine is one of the major determinants of

reaction specificity in PLP-dependent enzymes; however, several other factors such as

hydrogen bonding interactions, torsion and orientation of the cofactor, appear to be important

[7]. The unique environment provided by the apoprotein of a PLP-dependent enzyme drives

the catalytic power of the coenzyme so that the required reaction is optimized, while all the

Cystalysin: An Example of the Catalytic Versatility… 101

other possibilities are almost completely prevented. However, due to the large number of

alternatives, “mistakes” may occur. As a consequence, most PLP-enzymes are able to

catalyze side reactions which have a limited efficiency, but sometimes assume a

physiological meaning [1]. A schematic representation of the different reactions catalyzed by

PLP-dependent enzymes is shown in Figure 1.

Internal

aldimine

External

aldimine

Quinonoid

Quinonoid Quinonoid

Quinonoid

Quinonoid β,γ−unsatured

aldimine

α,β−unsatured

aldimine

H+

 from Cα

R from Cα CO2 from Cα

H+ H to C4’ +

 to Cα

X from Cγ

H+

 from Cβ

H+

 to Cγ

X from Cβ

R to Cβ

H+

 to Cα

H+ H to C4’ +

 to Cα

R to Cα

H+

 to Cα

H+

 to Cα

R to Cγ

β−Eliminases

Racemases

α−Synthases

Serine

hydroxymethyl

transferase

Transaminating

decarboxylases

Decarboxylases

β−Synthases

Aminotransferases

γ−Synthases

Figure 1. Schematic representation of the catalytic versatility of pyridoxal 5’-phosphate (PLP)

dependent enzymes. Each reaction begins with conversion of the internal to external aldimine. Covalent

modifications occurring at successive steps are indicated on the arrows connecting the intermediates.

Cystalysin is a PLP-dependent lyase which catalyzes the α,β-elimination of L-cysteine to

pyruvate, ammonia and sulfidric acid. The protein is produced by T.denticola, an oral

pathogen found at elevated concentrations in the gingival crevice of patients affected by

adulte periodontitis. T. denticola produces a large number of virulence factors including

several proteolytic and cytotoxic enzymes, required for bacterial growth in the periodontal

pocket and disease progression [8]. Cystalysin was identified in 1994, when Holt and

coworkers, while studying the hemolytic and hemoxidative properties of T. denticola, found

that both activities were dependent on a 45 KDa cell-associated protein encoded by the hly

gene [9]. After cloning of the gene, it was possible to demonstrate that the hemolysin is a

cysteine Cβ-Sγ lyase homologous to PLP-dependent aminotransferases [10]. Cystalysin is

able to interact with human red blood cells causing spikes and protrusion in the erythrocyte

membrane, and leading to the formation of irregular holes. Furthermore, the protein causes

the oxidation and sulfuration of hemoglobin to methemoglobin and sulfhemoglobin,

respectively [11]. Various studies have suggested that cystalysin induces haemolysis by a

novel mechanism, possibly dependent on its catalytic activity which determines production of

H2S. This compound is toxic for most cells and, by lysing erythrocytes, it allows the delivery

of many nutrition factors, including various amino acids and the iron of the haem [12].

Moreover, T. denticola belongs to a limited number of oral pathogens able to produce and

102 Barbara Cellini, Riccardo Montioli and Carla Borri Voltattorni

tolerate high concentrations (mM) of H2S found in periodontal disease pockets [13]. This

ability gives selective advantages to the bacterium allowing the formation of an ecological

niche in the periodontal pocket. Thus, the major function of cystalysin seems to be the

production of H2S and the protein can be regarded as a true PLP-dependent virulence factor

[12].

The crystal structure of cystalysin and cystalysin-L-aminoethoxyvinylglycine complex,

solved in 2000 by Krupka and coworkers, reveals that the protein belongs to Fold Type I or

L-aspartate aminotransferase family of PLP-dependent enzymes [14] (Figure 2). The protein

is a homodimer with 399 amino acids per subunit. Each monomer folds into two domains: i) a

large domain, consisting of residues 48-288 and carrying the PLP cofactor covalently bound

to Lys 238; ii) a small domain, consisting of the two terminal regions of the polypeptide

chain. In the centre of each cystalysin monomer, PLP is bound in a wide catalytic cleft

formed by both domains of one subunit and parts of the large domain of the other subunit.

The cofactor is bound by different types of interactions including the Schiff base linkage with

Lys 238 and ring-stacking interactions of the pyridine ring with the phenol ring of Tyr 123.

In addition, PLP is strongly anchored to the apoprotein through its phosphate group, which

forms six hydrogen bonds with protein residues and two hydrogen bonds with two water

molecules [14].

 

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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Environ. Microbiol., 69, 760-768.

Lechner, U.; Brodkorb, D.; Geyer, R.; Hause, G.; Härtig, C.; Auling, G.; Fayolle-Guichard,

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Lopes Ferreira, N.; Malandain, C.; Fayolle-Guichard, F. (2006). Enzymes and genes involved

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methylmalonyl-CoA mutase. Biochemistry, 38, 7999-8005.

Marsh, E. N. & Leadlay, P. F. (1989). Methylmalonyl-CoA mutase from Propionibacterium

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339-343.

Marsh, E. N.; McKie, N.; Davis, N. K.; Leadlay, P. F. (1989). Cloning and structural

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anaerobic bacterium WoG13 and methanogenic isobutyrate degradation by a defined

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Matthies, C.; Springer, N.; Ludwig, W.; Schink, B. (2000). Pelospora glutarica gen. nov., sp.

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the stereospecificity of the coenzyme B12-dependent isobutyryl-CoA mutase reaction. J.

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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.