TRIPASS XR تري باس

 

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.

REFERENCES

Allen, S. H. G.; Kellermeyer, R. W.; Stjernholm, R.; Wood, H. G. (1964). Purification and

properties of enzymes involved in the propionic acid fermentation. J. Bacteriol., 87, 171-

187.

Angelidaki, I. & Ahring, B. K. (1995). Isomerization of n- and i-butyrate in anaerobic

methanogenic systems. Antonie van Leeuwenhoek, 68, 285-291.

Banerjee, A.; Sharma, R.; Banerjee, U. C. (2002). The nitrile-degrading enzymes: current

status and future prospects. Appl. Microbiol. Biotechnol., 60, 33-44.

Birch, A.; Leiser, A.; Robinson, J. A. (1993). Cloning, sequencing, and expression of the

gene encoding methylmalonyl-coenzyme A mutase from Streptomyces cinnamonensis. J.

Bacteriol., 175, 3511-3519.

New Bacterial CoA-Carbonyl Mutases 95

Callaghan, A. V; Gieg, L. M.; Kropp, K. G.; Suflita, J. M.; Young, L. Y. (2006). Comparison

of mechanisms of alkane metabolism under sulfate-reducing conditions among two

bacterial isolates and a bacterial consortium. Appl. Environ. Microbiol., 72, 4274–4282.

Catelani, D.; Colombi, A.; Sorlini, C.; Treccani, V. (1977). Metabolism of quaternary carbon

compounds: 2,2-dimethylheptane and tertbutylbenzene. Appl. Environ. Microbiol., 34,

351-354.

Chen, Y. F.; Ng, W. J.; Yap, M. G. S. (1994). Performance of upflow anaerobic biofilter

process in pharmaceutical wastewater treatment. Resour. Conserv. Recycl., 11, 83-91.

Cherie Ligniere, C.; Montagnani, G.; Alberici, M.; Acerbi, D. (1987). Plasma and synovial

fluid concentrations of piroxicam during prolonged treatment with piroxicam pivalic

ester. Arzneimittelforschung, 37, 560-563.

Chisholm, M. S. (2000). Artificial glass—the versatility of poly(methyl methacrylate) from

its early exploitation to the new millennium. J. Chem. Edu., 77, 841-845.

Costa, E.; Perez, J.; Kreft, J.-U. (2006). Why is metabolic labour divided in nitrification?

Trends in Microbiol., 14, 213-219.

Cravo-Laureau, C.; Grossi, V.; Raphel, D.; Matheron, R.; Hirschler-Réa, A. (2005).

Anaerobic n-alkane metabolism by a sulfate-reducing bacterium, Desulfatibacillum

aliphaticivorans strain CV2803T

. Appl. Environ. Microbiol., 71, 3458-3467.

Czerkawski, J. W. (1976). The use of pivalic acid as a reference substance in measurements

of production of volatile fatty acids by rumen microorganisms. Br. J. Nutr., 36, 311-315.

Deodato, F.; Boenzi, S.; Santorelli, F. M.; Dionsisi-Vici, C. (2006). Methylmalonic and

propionic aciduria. Am. J. Med. Genet. C Semin. Med. Genet., 142, 104-112.

EPA - U. S. Environmental Protection Agency, Office of Water. Drinking water advisory:

consumer acceptability advice and health effects analysis on methyl tertiary butyl ether

(MTBE). EPA-822-F-97-008. Washington DC: U.S. Environmental Protection Agency;

1997.

Fayolle, F.; Vandecasteele, J.-P.; Monot, F. (2001). Microbial degradation and fate in the

environment of methyl tert-butyl ether and related fuel oxygenates. Appl. Microbiol.

Biotechnol., 56, 339-349.

Forslund, K.; Morant, M.; Jørgensen, B.; Olsen, C. E.; Asamizu, E.; Sato, S.; Tabata, S.; Bak,

S. (2004). Biosynthesis of the nitrile glucosides rhodiocyanoside A and D and the

cyanogenic glucosides lotaustralin and linamarin in Lotus japonicus. Plant Physiol., 135,

71-84.

François, A.; Mathis, H.; Godefroy, D.; Piveteau, P.; Fayolle, F.; Monot, F. (2002).

Biodegradation of methyl tert-butyl ether and other fuel oxygenates by a new strain,

Mycobacterium austroafricanum IFP 2012. Appl. Environ. Microbiol., 68, 2754-2762.

Gani, D.; O´Hagan, D.; Reynolds, K.; Robinson, J. A. (1985). Biosynthesis of the polyether

antibiotic monensin-A: stereochemical aspects of the incorporation and metabolism of

isobutyrate. J. Chem. Soc., Chem. Commun., 1002-1004.

Gruber, K. & Kratky, C. Methylmalonyl CoA mutase. In Messerschmidt, A.; Huber, R.;

Poulos, T.; Wieghardt, K., editors. Handbook of metalloproteins. Chichester: John

Wiley; 2001; 995-1009.

Henderson, R. F.; Sabourin, P. J.; Bechtold, W. E.; Steinberg, B.; Chang, L.-Y. (1993).

Disposition of inhaled isobutene in F344/N rats. Toxicol. Appl. Pharmacol., 123, 50-61.

96 Thore Rohwerder and Roland H. Müller

Janssen, D. B.; Dinkla, I. J. T.; Poelarends, G. J.; Terpstra, P. (2005). Bacterial degradation of

xenobiotic compounds: evolution and distribution of novel enzyme activities. Environ.

Microbiol., 7, 1868-1882.

Kniemeyer, O.; Fischer, T.; Wilkes, H.; Glöckner, F. O.; Widdel, F. (2003). Anaerobic

degradation of ethylbenzene by a new type of marine sulfate-reducing bacterium. Appl.

Environ. Microbiol., 69, 760-768.

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

F.; Piveteau, P.; Müller, R. H.; Rohwerder, T. (2007). Aquincola tertiaricarbonis gen.

nov., sp. nov., a tertiary butyl moieties degrading bacterium. Int. J. Syst. Evol.

Microbiol., 57, 1295-1303.

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

in the aerobic biodegradation of MTBE. Appl. Microbiol. Biotechnol, 72, 252-262.

Mancia, F.; Smith, G. A.; Evans, P. R. (1999). Crystal structure of substrate complexes of

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

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

shermanii. Evidence for the presence of two masked cysteine residues. Biochem. J., 260,

339-343.

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

characterization of the genes coding for adenosylcobalamin-dependent methylmalonylCoA mutase from Propionibacterium shermanii. Biochem. J., 260, 345-352.

Martens, J.-H.; Barg, H.; Warren, M. J.; Jahn, D. (2002). Microbial production of vitamin

B12. Appl. Microbiol. Biotechnol., 58, 275-285.

Matthies, C. & Schink, B. (1992). Reciprocal isomerization of butyrate by the striclty

anaerobic bacterium WoG13 and methanogenic isobutyrate degradation by a defined

triculture. Appl. Environ. Microbiol., 58, 1435-1439.

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

nov., a glutarate-fermenting, strictly anaerobic, spore-forming bacterium. Int. J. Syst.

Evol. Microbiol., 50, 645-648.

Moore, B. S.; Eisenberg, R.; Weber, C.; Bridges, A.; Nanz, D.; Robinson, J. A. (1995). On

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

Am. Chem. Soc., 117, 11285-11291.

Moran, M. J.; Zogorski, J. S.; Squillace, P. J. (2005). MTBE and gasoline hydrocarbons in

ground water of the United States. Ground Water, 43, 615-627.

Müller, R. H.; Rohwerder, T.; Harms, H. (2007). Carbon conversion efficiency and limits of

productive bacterial degradation of methyl tert-butyl ether (MTBE) and related

compounds. Appl. Environ. Microbiol., 73,1783-1791.

Oude Elferink, S. J. W. H.; Lens, P. N. L.; Dijkema, C.; Stams, A. J. M. (1996).

Isomerization of butyrate to isobutyrate by Desulforhabdus amnigenus. FEMS Microbiol.

Lett., 142, 237-241.

Padmakumar, R. & Banerjee, R. A. (1995). Carbon-skeleton walk: a novel double

rearrangement of glutaryl-CoA catalyzed by the human methylmalonyl-CoA mutase.

Biofactors, 5, 83-86.

New Bacterial CoA-Carbonyl Mutases 97

Perri, K. L. The effectiveness of multiple redox treatment strategies on the treatability of a

high strength industrial wastewater. Masters´s thesis. Blacksburg: Virginia Polytechnic

Institute and State University; 1997.

Pfeiffer, T. & Bonhoeffer, S. (2002). Evolutionary consequences of tradeoffs between yield

and rate of ATP production. Z. Phys. Chem., 216, 51-63.

Probian, C.; Wülfing, A.; Harder, J. (2003). Anaerobic mineralization of quaternary carbon

atoms: isolation of denitrifying bacteria on pivalic acid (2,2-dimethylpropionic acid).

Appl. Environ. Microbiol., 69, 1866-1870.

Ratnatilleke, A.; Vrijbloed, J. W.; Robinson, J. A. (1999). Cloning and sequencing of the

coenzyme B12-binding domain of isobutyryl-CoA mutase from Streptomyces

cinnamonensis, reconstitution of mutase activity, and characterization of the recombinant

enzyme produced in Escherichia coli. J. Biol. Chem., 274, 31679-31685.

Raut, N. M.; Jaison, P. G.; Aggarwal, S. K. (2002). Comparative evaluation of three αhydroxycarboxylic acids for the separation of lanthanides by dynamically modified

reversed-phase high-performance liquid chromatography. J. Chromatogr. A, 959, 163-

172.

Rétey, J.; Smith, E. H.; Zagalak, B. (1978). Investigation of the mechanism of the

methylmalonyl-CoA mutase reaction with substrate analogue: ethylmalonyl-CoA. Eur. J.

Biochem., 83, 437-451.

Reynolds, K. A.; O’Hagan, D.; Gani, D.; Robinson, J. A. (1988). Butyrate metabolism in

streptomycetes. Characterization of an intramolecular interchange rearrangement linking

isobutyrate and butyrate in Streptomyces cinnamonensis. J. Chem. Soc. Perkin Trans. I,

3195–3207.

Rohwerder, T.; Breuer, U.; Benndorf, D.; Lechner, U.; Müller, R. H. (2006). The alkyl tertbutyl ether intermediate 2-hydroxyisobutyrate is degraded via a novel cobalamindependent mutase pathway. Appl. Environ. Microbiol., 72, 1428-4135.

Sauber, K.; Aretz, W.; Meiwes, J.; Wollmann, T. (1996). A new esterase for the cleavage of

pivalic acid-containing prodrug esters of cephalosporins. Enzyme Microb. Technol., 19,

15-19.

Schiffman, S. S.; Bennett, J. L.; Raymer, J. H. (2001). Quantification of odors and odorants

from swine operations in North Carolina. Agric. For. Meteorol., 108, 213-240.

Schmidt, T. C.; Morgenroth, E.; Schirmer, M.; Effenberger, M.; Haderlein, S. B. Use and

occurrence of fuel oxygenates in Europe. In Diaz, A. F. & Drogos, D. L., editors.

Oxygenates in gasoline: environmental aspects. Washington DC: ACS; 2002; 58-79.

Smith, M. & Essenberg, C. Pivalic Acid Pathway Map (Anaerobic) [online]. 2006 April 20.

Available from: http://umbbd.msi.umn.edu/pva/pva_map.html.

Sekiguchi, Y.; Kamagata, Y.; Nakamura, K.; Ohashi, A.; Harada, H. (2000).

Syntrophothermus lipocalidus gen. nov., sp. nov., a novel thermophilc, syntrophic, fattyacid-oxidizing anaerobe which utilizes isobutyrate. Int. J. Syst. Evol. Microbiol., 50, 771-

779.