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+---Topic: Evolution of multiple-parts-required pathways started by niiicholas

Posted by: niiicholas on Dec. 19 2002,00:04

This is a big enough topic to deserve a thread separate from the origin of information or the origin of particular systems.

Short version: there is lots of evidence that multiple-parts-required metabolic pathways have originated via known evolutionary processes, in human and even lab lifetimes.

Here is a synthesis article I just came across:


Curr Opin Struct Biol 2002 Jun;12(3):374-82
< Pathway evolution, structurally speaking. >

Rison SC, Thornton JM.

Department of Biochemistry and Molecular Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, UK.

Small-molecule metabolism forms the core of the metabolic processes of all living organisms. As early as 1945, possible mechanisms for the evolution of such a complex metabolic system were considered. The problem is to explain the appearance and development of a highly regulated complex network of interacting proteins and substrates from a limited structural and functional repertoire. By permitting the co-analysis of phylogeny and metabolism, the combined exploitation of pathway and structural databases, as well as the use of multiple-sequence alignment search algorithms, sheds light on this problem. Much of the current research suggests a chemistry-driven 'patchwork' model of pathway evolution, but other mechanisms may play a role. In the future, as metabolic structure and sequence space are further explored, it should become easier to trace the finer details of pathway development and understand how complexity has evolved.


Then of course we have:


Trends in Biochemical Sciences
Volume 25, Issue 6, 1 June 2000, Pages 261-265
< Evolution of a metabolic pathway for degradation of a toxic xenobiotic: the patchwork approach >

Shelley D. Copley

The pathway for degradation of the xenobiotic pesticide pentachlorophenol in Sphingomonas chlorophenolica probably evolved in the past few decades by the recruitment of enzymes from two other catabolic pathways. The first and third enzymes in the pathway, pentachlorophenol hydroxylase and 2,6-dichlorohydroquinone dioxygenase, may have originated from enzymes in a pathway for degradation of a naturally occurring chlorinated phenol. The second enzyme, a reductive dehalogenase, may have evolved from a maleylacetoacetate isomerase normally involved in degradation of tyrosine. This apparently recently assembled pathway does not function very well: pentachlorophenol hydroxylase is quite slow, and tetrachlorohydroquinone dehalogenase is subject to severe substrate inhibition.


An important update:


J Bacteriol 2003 Jan;185(1):302-10
< A Previously Unrecognized Step in Pentachlorophenol Degradation in Sphingobium chlorophenolicum Is Catalyzed by Tetrachlorobenzoquinone Reductase (PcpD). >

Dai M, Rogers JB, Warner JR, Copley SD.

The first step in the pentachlorophenol (PCP) degradation pathway in Sphingobium chlorophenolicum has been believed for more than a decade to be conversion of PCP to tetrachlorohydroquinone. We show here that PCP is actually converted to tetrachlorobenzoquinone, which is subsequently reduced to tetrachlorohydroquinone by PcpD, a protein that had previously been suggested to be a PCP hydroxylase reductase. pcpD is immediately downstream of pcpB, the gene encoding PCP hydroxylase (PCP monooxygenase). Expression of PcpD is induced in the presence of PCP. A mutant strain lacking functional PcpD has an impaired ability to remove PCP from the medium. In contrast, the mutant strain removes tetrachlorophenol from the medium at the same rate as does the wild-type strain. These data suggest that PcpD catalyzes a step necessary for degradation of PCP, but not for degradation of tetrachlorophenol. Based upon the known mechanisms of flavin monooxygenases such as PCP hydroxylase, hydroxylation of PCP should produce tetrachlorobenzoquinone, while hydroxylation of tetrachlorophenol should produce tetrachlorohydroquinone. Thus, we proposed and verified experimentally that PcpD is a tetrachlorobenzoquinone reductase that catalyzes the NADPH-dependent reduction of tetrachlorobenzoquinone to tetrachlorohydroquinone.


Pentachlorophenol (PCP) is a widely used and highly toxic wood preservative. It was first introduced as a pesticide in 1936 (7) and is not known to be a natural product. Despite its recent introduction into the environment and its high toxicity, several strains of Sphingobium chlorophenolicum (previously Sphingomonas chlorophenolica) (24) that can mineralize PCP have been identified. The best studied of these are strains ATCC 39723 (19), RA-2 (23), and UG30 (6). It appears that S. chlorophenolicum has assembled a new metabolic pathway capable of converting this anthropogenic compound into a recognizable metabolite. Our previous studies suggest that this pathway has been assembled by patching together enzymes from at least two different metabolic pathways (8). PCP hydroxylase (PCP monooxygenase; EC and 2,6-dichlorohydroquinone dioxygenase may have originated from enzymes that hydroxylated a naturally occurring chlorinated phenol and then cleaved the resulting hydroquinone. Tetrachlorohydroquinone (TCHQ) dehalogenase appears to have originated from a glutathione-dependent double bond isomerase such as maleylacetoacetate isomerase or maleylpyruvate isomerase (which are involved in degradation of tyrosine and benzoate, respectively) (2). If this pathway has evolved recently in response to the introduction of PCP into the environment, then it would not be expected to perform at the high level characteristic of pathways that have evolved over periods of millions or billions of years. Indeed, the PCP degradation pathway shows signs of immaturity in several respects. First, PCP hydroxylase, the first enzyme in the pathway, is very inefficient in vitro (P. M. Kiefer and S. D. Copley, unpublished data), and appears to severely limit the flux of PCP through the pathway in vivo (17). Second, TCHQ dehalogenase is profoundly inhibited by its aromatic substrate (K. Anandarajah, P. M. Kiefer, and S. D. Copley, unpublished data). Third, TCHQ dehalogenase expression is not regulated in tandem with the other known enzymes in the pathway but is apparently constitutive (21). All of these findings are consistent with the idea that the PCP degradation pathway has been patched together rather recently and has not been fine-tuned to perform as effectively as do most bacterial metabolic pathways.

The gene encoding PCP hydroxylase (pcpB) is immediately upstream of two additional genes. pcpR encodes a regulatory protein that responds to PCP (5). pcpD, which is immediately downstream of pcpB, resembles genes for the reductase components of two-component oxygenases, some of which hydroxylate aromatic compounds. Based upon this resemblance, it has been proposed that PcpD is a reductase that facilitates the hydroxylation of PCP by PCP hydroxylase (19), and the annotation of PcpD in GenBank states that it is PCP 4-monooxygenase reductase. We suspected that this assignment was incorrect because PCP hydroxylase is a flavin monooxygenase, and such enzymes do not generally require reductases. Consequently, we undertook studies to determine whether PcpD is required for degradation of PCP. We find that transcription of pcpD is induced by PCP, as previously reported for pcpA (29) and pcpB (20). A mutant strain in which PcpD has been knocked out is able to remove PCP from the medium when it is present at low concentrations, but not when it is present at high concentrations. In contrast, the knockout strain can remove tetrachlorophenol (TCP) from the medium as well as the wild-type strain, even at high concentrations. These results suggest that PcpD may catalyze a step that is critical for degradation of PCP but not TCP and therefore must involve the chlorine at the 4 position of PCP. Based upon the expected mechanism of the hydroxylase reaction, the sequence of PcpD, and our experimental results, we propose that PcpD is a tetrachlorobenzoquinone (TCBQ) reductase required for degradation of PCP but not TCP.


Posted by: niiicholas on July 05 2003,17:37

More fun with atrazine and related compounds:


< >

Biochemistry, 40 (43), 12747 -12753, 2001. 10.1021/bi011293r S0006-2960(01)01293-4
Web Release Date: October 3, 2001

Copyright © 2001 American Chemical Society
Rapid Evolution of Bacterial Catabolic Enzymes: A Case Study with Atrazine Chlorohydrolase

Jennifer L. Seffernick and Lawrence P. Wackett*

This review discusses examples in which it is possible to sift through the complexity of the biosphere to find related enzymes which display distinct functions. The clearest example to date is atrazine chlorohydrolase, an enzyme which is shown to have evolved for the function of catabolizing atrazine. More than 2 billion pounds of the herbicide atrazine have been applied to soils globally, and this has provided selective pressure for the evolution of new metabolism. The amino acid sequence of atrazine chlorohydrolase is shown to be 98% identical with that of melamine deaminase, an enzyme that catalyzes deamination reactions. The chlorohydrolase is shown to be firmly linked with a major amidohydrolase protein superfamily.

Atrazine Catabolism and the Amidohydrolase Superfamily
The chlorinated herbicide atrazine was once considered to be poorly biodegraded in soils. The major metabolites detected in soils and groundwaters suggested that the herbicide underwent nonspecific oxidative dealkylation reactions (Figure 1). A cytochrome P450 monooxygenase from Rhodococcus strains TE1, N186/221, and B30 was subsequently discovered to catalyze this reaction (20-24). The bacterial cytochrome P450 was shown to degrade other herbicides structurally unrelated to atrazine and is likely functioning as a nonspecific oxygenative catalyst rather than an enzyme that has evolved specifically to catabolize atrazine. Starting in 1993, however, numerous bacteria were ascertained to initiate atrazine metabolism via a hydrolytic dechlorination reaction (Figure 2). More recently, the genes encoding the chlorohydrolase have been shown to be essentially identical in different genera of bacteria independently isolated from four continents by different researchers (25). This suggests that the ability to dechlorinate atrazine arose since the introduction of atrazine and that this phenotype spread quickly around the globe.

The enzymes responsible for the first three steps of the atrazine dehalogenation pathway were initially identified in Pseudomonas sp. strain ADP (Figure 2). The enzymes that catalyze these steps are atrazine chlorohydrolase (AtzA, EC, hydroxyatrazine ethylaminohydrolase (AtzB, EC, and N-isopropylammelide N-isopropylaminohydrolase (AtzC, EC, respectively. Sequence comparisons revealed that all three enzymes belong to the amidohydrolase superfamily (26). Amidohydrolase superfamily members for which structures are defined have an ()8 barrel structure (27, 28). Moreover, they share conserved features of the reaction mechanism in which one or two divalent metals are coordinated by the enzyme and serve to activate water for nucleophilic attack on the respective substrate. The amino acids serving as metal ligands are maintained across the superfamily. The majority of reactions catalyzed by the superfamily involve the hydrolytic removal of amino groups from purine and pyrimidine rings, or amide bond hydrolysis reactions (Figure 3). The former reactions are represented by enzymes such as adenosine deaminase. The latter are illustrated by urease and cyclic amidases such as hydantoinase.

Recent studies have expanded the range of reactions that are known to be catalyzed by amidohydrolase superfamily members (Figure 3). Some of the existing enzymes catabolize synthetic organic compounds (Table 1). Phosphotriesterase, for instance, catalyzes the cleavage of a phosphorus-oxygen bond of the pesticide parathion (29). It has been speculated that the true substrate for phosphotriesterase from Pseudomonas dismuta is yet to be discovered. But it is also plausible that the enzyme has evolved under selective pressure to hydrolyze phosphotriester insecticides since their introduction some decades ago.


Other data support the view that the Pseudomonas AtzA evolved under selective pressure and was maintained in soil microbial populations to metabolize s-triazine herbicides. The atzA gene was not found in randomly chosen laboratory strains but was detected in most bacteria recently isolated for their ability to metabolize atrazine (25). It is present with other genes, atzB and atzC, which encode enzymes that metabolize the AtzA reaction product in Pseudomonas sp. ADP Ralstonia, Alcaligenes, and Agrobacterium strains (Figure 2) (25). The atrazine catabolism genes are found on large catabolic plasmids in those same strains (42).

Melamine Deaminase and s-Triazine Hydrolase

Perhaps the best evidence that atzA is a recently evolved gene derives from its relationship with genes identified for the catabolism of melamine, or 2,4,6-triamino-1,3,5-triaizine. Melamine is an industrial product used since the early 1900s. Melamine was considered nonbiodegradable in the 1930s but was then reclassified as slightly biodegradable in the 1960s when atrazine was first introduced (43). Today, it is considered to be readily biodegradable in soil. Among the bacteria that metabolize melamine is Acidovorax avenae citrulli 12227 (formerly Pseudomonas sp. strain NRRL B-12227) (44). The first two metabolic reactions are sequential hydrolytic deamination reactions catalyzed by the same enzyme, melamine deaminase (TriA). The triA gene has recently been cloned and sequenced. The protein shows a remarkable identity to atrazine chlorohydrolase from Pseudomonas sp. ADP; it is the same in 466 of 475 amino acids (Figure 4) (45). It is also unusual that the nine nucleotide differences between triA and atzA give rise to these nine amino acid changes. The small number of changes and the absence of silent mutations are consistent with an intense selective pressure operating over a short evolutionary time period (46, 47). The kcat/Km of atrazine chlorohydrolase with atrazine is 1.5 × 104 s-1 M-1 per subunit. In our most recent study, the deamination activity of this enzyme was found to be undetectable (48). Melamine deaminase, however, exhibits the opposite specificity. It catalyzes deamination reactions at rates comparable to dechlorination rates of atrazine chlorohydrolase. Moreover, it shows dechorination activity 2 orders of magnitude lower than the deamination activity with comparable triazine substrates. In total, these data suggest that the nine amino acid changes represent a short evolutionary trajectory between the two activities.


The sequence of a related amidohydrolase superfamily member, s-triazine hydrolase or TrzA (49), is 41 and 42% identical with the sequences of atrazine chlorohydrolase and melamine deaminase, respectively. It catalyzes both deamination and dechlorination reactions. TrzA catalyzes the deamination of nonalkylated triazines such as melamine and the dechlorination of mono-N-alkylated triazines. The kcat for deamination of melamine is 243 s-1, while that for the dechlorination of desisopropylatrazine is 2.2 s-1. This is an approximately 100 times greater preference for demination over dechlorination and is consistent with the enzyme acting physiologically as a deaminase with a fortuitous dechlorination activity. This is not surprising given that chloride displacement is more facile, and adenosine deaminase is known to catalyze fortuitous halopurine dehalogenation. That TriA and AtzA discriminate between chloro and amino substrates so well despite their sequences being 98% identical is remarkable.

DNA Shuffling
It is possible that fewer than nine amino acid changes are required to interconvert melamine deaminase and atrazine chlorohydrolase activities. There are 510 possible site-directed mutants bridging the two, a large set to generate, sequence, purify, and assay. In this context, DNA shuffling was conducted and the clonal variants were screened against a chemical library of substrates using high-throughput mass spectrometry (48). The chemical library of 15 substrates varied the leaving group and the side chains (Figure 5). Mutant enzymes were obtained that varied with respect to their activities against the different substrates. The sequences of daughter enzymes exhibiting the greatest activity for hydrolysis of atrazine analogues are displayed in Table 2. The activities of the shuffled clones were normalized to the activity of each parental enzyme. The clone with the best dechlorination activity was 1.4 times as fast as atrazine chlorohydrolase, and the clone with the best deamination activity was 3.6 times better than melamine deaminase. The small increases observed in activity upon shuffling suggest that atrazine chlorohydrolase and melamine deaminase have among the most optimal sequences for dechlorination and deamination activities, respectively.

It is also of potential evolutionary significance that shuffled mutants were obtained with 80-fold enhanced activities with substrates containing methyl thioether and methoxy substituents. These represent the commercially relevant herbicides ametryn and atraton, respectively. An enzyme purified from a Nocardioides sp. was shown to hydrolyze ametryn, but it was not tested with atraton or other methoxy-functionalized herbicides (50). DNA from the Nocardioides sp. did not hybridize to an atzA probe, suggesting that the enzyme does not closely resemble atrazine chlorohydrolase from Pseudomonas sp. ADP. However, the data in Table 2 suggest that enzymes capable of metabolizing ametryn, atraton, and related triazine herbicides could be derived from triA or closely homologous genes in nature.

With respect to the sequences that favor dechlorination versus deamination, the data show a trend in that residue 328 appears to largely control leaving group specificity. Asn328 tracks with narrow specificity enzymes that largely catalyze dechlorination. Asp328 tracks with broader specificity enzymes which catalyze deamination and the displacement of -NCH3, -OCH3, and -SCH3 groups. The hypothesis that this residue is crucial to the observed specificity difference between melamine deaminase and atrazine chlorohydrolase is currently being addressed with site-directed mutagenesis studies.

Nature must continually fine-tune enzyme substrate specificities and reaction rates over time under the aegis of biological need, usually called selective evolutionary pressure. This enzyme variability is particularly marked with soil bacteria due to their enormous numbers, large evolutionary span of 3.6 billion years, rapid reproductive rates, and great competition for scarce nutrient resources. Enzyme plasticity is important in this context, but this confounds genome annotation efforts where gene function is assigned on the basis of finding the homologue with the most identical sequence. As discussed here, enzymes with sequences that are 98% identical can catalyze different reactions. It will be imperative to flesh out a broader range of microbial enzymatic reactions, particularly for microbial catabolic enzymes where the diversity of enzymes will likely be great.


Posted by: niiicholas on July 14 2003,00:30

< >

Whoa, Nelson just admitted that multiple-parts required systems can evolve rapidly by cooption of parts.  Which is what we've been arguing all along:


Another great example of this might be the 2,4 DNT pathway. The first operon is taken from the naphthalene degradation pathway, the second operon is taken from distantly related chlorophenol and nitrophenol pathways. There are others but finding out where they came from would just be a guess. However, it shows just how bacteria when faced with something completely new have the tools to construct something just as new in order to overcome the challenge.

It looks like this paper may have convinced him that IC isn't all its cracked up to be:


J Bacteriol. 2002 Aug;184(15):4219-32.

Erratum in:
J Bacteriol 2002 Nov;184(21):6084.
< Origins of the 2,4-dinitrotoluene pathway. >

Johnson GR, Jain RK, Spain JC.

Air Force Research Laboratory, U.S. Air Force, Tyndall Air Force Base, Florida 32403, USA.

The degradation of synthetic compounds requires bacteria to recruit and adapt enzymes from pathways for naturally occurring compounds. Previous work defined the steps in 2,4-dinitrotoluene (2,4-DNT) metabolism through the ring fission reaction. The results presented here characterize subsequent steps in the pathway that yield the central metabolic intermediates pyruvate and propionyl coenzyme A (CoA). The genes encoding the degradative pathway were identified within a 27-kb region of DNA cloned from Burkholderia cepacia R34, a strain that grows using 2,4-DNT as a sole carbon, energy, and nitrogen source. Genes for the lower pathway in 2,4-DNT degradation were found downstream from dntD, the gene encoding the extradiol ring fission enzyme of the pathway. The region includes genes encoding a CoA-dependent methylmalonate semialdehyde dehydrogenase (dntE), a putative NADH-dependent dehydrogenase (ORF13), and a bifunctional isomerase/hydrolase (dntG). Results from analysis of the gene sequence, reverse transcriptase PCR, and enzyme assays indicated that dntD dntE ORF13 dntG composes an operon that encodes the lower pathway. Additional genes that were uncovered encode the 2,4-DNT dioxygenase (dntAaAbAcAd), methylnitrocatechol monooxygenase (dntB), a putative LysR-type transcriptional (ORF12) regulator, an intradiol ring cleavage enzyme (ORF3), a maleylacetate reductase (ORF10), a complete ABC transport complex (ORF5 to ORF8), a putative methyl-accepting chemoreceptor protein (ORF11), and remnants from two transposable elements. Some of the additional gene products might play as-yet-undefined roles in 2,4-DNT degradation; others appear to remain from recruitment of the neighboring genes. The presence of the transposon remnants and vestigial genes suggests that the pathway for 2,4-DNT degradation evolved relatively recently because the extraneous elements have not been eliminated from the region.


Or perhaps it was this more recent one that just turned up in 'related articles':


Appl Microbiol Biotechnol. 2003 May 15 [Epub ahead of print].  
< Evolution of catabolic pathways for synthetic compounds: bacterial pathways for degradation of 2,4-dinitrotoluene and nitrobenzene. >

Johnson GR, Spain JC.

Air Force Research Laboratory, United States Air Force, FL 32403, Tyndall Air Force Base, USA.

The pathways for 2,4-dinitrotoluene (2,4-DNT) and nitrobenzene offer fine illustrations of how the ability to assimilate new carbon sources evolves in bacteria. Studies of the degradation pathways provide insight about two principal strategies for overcoming the metabolic block imposed by nitro- substituents on aromatic compounds. The 2,4-DNT pathway uses novel oxygenases for oxidative denitration and subsequent ring-fission. The nitrobenzene pathway links facile reduction of the nitro- substituent, a novel mutase enzyme, and a conserved operon encoding aminophenol degradation for mineralization of nitrobenzene. Molecular genetic analysis with comparative biochemistry reveals how the pathways were assembled in response to the recent appearance of the two synthetic chemicals in the biosphere.


Imagine that, bacteria naturally evolving ways to break down human-designed chemicals they've never seen before...

Additional articles:


Curr Opin Biotechnol. 2003 Jun;14(3):262-9.  
< The role of mobile genetic elements in bacterial adaptation to xenobiotic organic compounds. >

Top EM, Springael D.

Department of Biological Sciences, 347 Life Sciences Building South, University of Idaho, 83844-3051, Moscow ID, USA

Retrospective studies clearly indicate that mobile genetic elements (MGEs) play a major role in the in situ spread and even de novo construction of catabolic pathways in bacteria, allowing bacterial communities to rapidly adapt to new xenobiotics. The construction of novel pathways seems to occur by an assembly process that involves horizontal gene transfer: different appropriate genes or gene modules that encode different parts of the novel pathway are recruited from phylogenetically related or distant hosts into one single host. Direct evidence for the importance of catabolic MGEs in bacterial adaptation to xenobiotics stems from observed correlations between catabolic gene transfer and accelerated biodegradation in several habitats and from studies that monitor catabolic MGEs in polluted sites.



Trends Biochem Sci. 2003 Jun;28(6):336-41.

< Metabolites: a helping hand for pathway evolution? >

Schmidt S, Sunyaev S, Bork P, Dandekar T.

European Molecular Biology Laboratory Heidelberg, Postfach 102209, D-69012, Heidelberg, Germany

The evolution of enzymes and pathways is under debate. Recent studies show that recruitment of single enzymes from different pathways could be the driving force for pathway evolution. Other mechanisms of evolution, such as pathway duplication, enzyme specialization, de novo invention of pathways or retro-evolution of pathways, appear to be less abundant. Twenty percent of enzyme superfamilies are quite variable, not only in changing reaction chemistry or metabolite type but in changing both at the same time. These variable superfamilies account for nearly half of all known reactions. The most frequently occurring metabolites provide a helping hand for such changes because they can be accommodated by many enzyme superfamilies. Thus, a picture is emerging in which new pathways are evolving from central metabolites by preference, thereby keeping the overall topology of the metabolic network.


Posted by: niiicholas on Nov. 21 2003,18:12

New articles.  References 6+ additional evolution-of-multiple-part-pathways-for-toxin-degradation examples in other articles.


Appl Microbiol Biotechnol. 2003 Aug;62(2-3):110-23.
< Evolution of catabolic pathways for synthetic compounds: bacterial pathways for degradation of 2,4-dinitrotoluene and nitrobenzene. >

Johnson GR, Spain JC.

Air Force Research Laboratory, United States Air Force, Tyndall Air Force Base, FL 32403, USA.

The pathways for 2,4-dinitrotoluene (2,4-DNT) and nitrobenzene offer fine illustrations of how the ability to assimilate new carbon sources evolves in bacteria. Studies of the degradation pathways provide insight about two principal strategies for overcoming the metabolic block imposed by nitro- substituents on aromatic compounds. The 2,4-DNT pathway uses novel oxygenases for oxidative denitration and subsequent ring-fission. The nitrobenzene pathway links facile reduction of the nitro- substituent, a novel mutase enzyme, and a conserved operon encoding aminophenol degradation for mineralization of nitrobenzene. Molecular genetic analysis with comparative biochemistry reveals how the pathways were assembled in response to the recent appearance of the two synthetic chemicals in the biosphere.



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