Shelley D. Copley

Evolution of a metabolic pathway for degradation of a toxic xenobiotic: the patchwork approach

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.

Moonlighting is mainstream: Paradigm adjustment required

Moonlighting – the performance of more than one function by a single protein – is becoming recognized as a common phenomenon with important implications for systems biology and human health. The different functions of a moonlighting protein may use different regions of the protein structure, or alternative structures that occur due to post‐translational modifications and/or differences in binding partners. Often the different functions of moonlighting proteins are used at different times or in different places. The existence of moonlighting functions complicates efforts to understand metabolic and regulatory networks, as well as physiological and pathological processes in organisms. Because moonlighting functions can play important roles in disease processes, an improved understanding of moonlighting proteins will provide new opportunities for pharmacological manipulations that specifically target a function involved in pathology while sparing physiologically important functions.

Genome Shuffling Improves Degradation of the Anthropogenic Pesticide Pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723

ABSTRACT Pentachlorophenol (PCP), a highly toxic anthropogenic pesticide, can be mineralized by Sphingobium chlorophenolicum, a gram-negative bacterium isolated from PCP-contaminated soil. However, degradation of PCP is slow and S. chlorophenolicum cannot tolerate high levels of PCP. We have used genome shuffling to improve the degradation of PCP by S. chlorophenolicum. We have obtained several strains that degrade PCP faster and tolerate higher levels of PCP than the wild-type strain. Several strains obtained after the third round of shuffling can grow on one-quarter-strength tryptic soy broth plates containing 6 to 8 mM PCP, while the original strain cannot grow in the presence of PCP at concentrations higher than 0.6 mM. Some of the mutants are able to completely degrade 3 mM PCP in one-quarter-strength tryptic soy broth, whereas no degradation can be achieved by the wild-type strain. Analysis of several improved strains suggests that the improved phenotypes are due to various combinations of mutations leading to an enhanced growth rate, constitutive expression of the PCP degradation genes, and enhanced resistance to the toxicity of PCP and its metabolites.

Evolution of efficient pathways for degradation of anthropogenic chemicals

Anthropogenic compounds used as pesticides, solvents and explosives often persist in the environment and can cause toxicity to humans and wildlife. The persistence of anthropogenic compounds is due to their recent introduction into the environment; microbes in soil and water have had relatively little time to evolve efficient mechanisms for degradation of these new compounds. Some anthropogenic compounds are easily degraded, whereas others are degraded very slowly or only partially, leading to accumulation of toxic products. This review examines the factors that affect the ability of microbes to degrade anthropogenic compounds and the mechanisms by which new pathways emerge in nature. New approaches for engineering microbes with enhanced degradative abilities include assembly of pathways using enzymes from multiple organisms, directed evolution of inefficient enzymes, and genome shuffling to improve microbial fitness under the challenging conditions posed by contaminated environments.

Lateral gene transfer and parallel evolution in the history of glutathione biosynthesis genes

BackgroundGlutathione is found primarily in eukaryotes and in Gram-negative bacteria. It has been proposed that eukaryotes acquired the genes for glutathione biosynthesis from the alpha-proteobacterial progenitor of mitochondria. To evaluate this, we have used bioinformatics to analyze sequences of the biosynthetic enzymes γ-glutamylcysteine ligase and glutathione synthetase.Resultsγ-Glutamylcysteine ligase sequences fall into three groups: sequences primarily from gamma-proteobacteria; sequences from non-plant eukaryotes; and sequences primarily from alpha-proteobacteria and plants. Although pairwise sequence identities between groups are insignificant, conserved sequence motifs are found, suggesting that the proteins are distantly related. The data suggest numerous examples of lateral gene transfer, including a transfer from an alpha-proteobacterium to a plant. Glutathione synthetase sequences fall into two distinct groups: bacterial and eukaryotic. Proteins in both groups have a common structural fold, but the sequences are so divergent that it is uncertain whether these proteins are homologous or arose by convergent evolution.ConclusionsThe evolutionary history of the glutathione biosynthesis genes is more complex than anticipated. Our analysis suggests that the two genes in the pathway were acquired independently. The gene for γ-glutamylcysteine ligase most probably arose in cyanobacteria and was transferred to other bacteria, eukaryotes and at least one archaeon, although other scenarios cannot be ruled out. Because of high divergence in the sequences, the data neither support nor refute the hypothesis that the eukaryotic gene comes from a mitochondrial progenitor. After acquiring γ-glutamylcysteine ligase, eukaryotes and most bacteria apparently recruited a protein with the ATP-grasp superfamily structural fold to catalyze synthesis of glutathione from γ-glutamylcysteine and glycine. The eukaryotic glutathione synthetase did not evolve directly from the bacterial glutathione synthetase.

Three serendipitous pathways in E. coli can bypass a block in pyridoxal-5′-phosphate synthesis

Bacterial genomes encode hundreds to thousands of enzymes, most of which are specialized for particular functions. However, most enzymes have inefficient promiscuous activities, as well, that generally serve no purpose. Promiscuous reactions can be patched together to form multistep metabolic pathways. Mutations that increase expression or activity of enzymes in such serendipitous pathways can elevate flux through the pathway to a physiologically significant level. In this study, we describe the discovery of three serendipitous pathways that allow synthesis of pyridoxal‐5′‐phosphate (PLP) in a strain of E. coli that lacks 4‐phosphoerythronate (4PE) dehydrogenase (PdxB) when one of seven different genes is overexpressed. We have characterized one of these pathways in detail. This pathway diverts material from serine biosynthesis and generates an intermediate in the normal PLP synthesis pathway downstream of the block caused by lack of PdxB. Steps in the pathway are catalyzed by a protein of unknown function, a broad‐specificity enzyme whose physiological role is unknown, and a promiscuous activity of an enzyme that normally serves another function. One step in the pathway may be non‐enzymatic.

A Previously Unrecognized Step in Pentachlorophenol Degradation in Sphingobium chlorophenolicum Is Catalyzed by Tetrachlorobenzoquinone Reductase (PcpD)

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.

Microbial dehalogenases: enzymes recruited to convert xenobiotic substrates

Microbial dehalogenases are involved in the biodegradation of many important chlorinated pollutants. Some recent studies of haloalkane dehalogenase, dichloromethane dehalogenase, tetrachlorohydroquinone dehalogenase and perchloroethylene and trichloroethylene reductive dehalogenases have addressed the issue of recruitment and adaptation of proteins to dehalogenate novel substrates.

A compromise required by gene sharing enables survival: Implications for evolution of new enzyme activities.

Evolution of new enzymatic activities is believed to require a period of gene sharing in which a single enzyme must serve both its original function and a new function that has become advantageous to the organism. Subsequent gene duplication allows one copy to maintain the original function, while the other diverges to optimize the new function. The physiological impact of gene sharing and the constraints imposed by the need to maintain the original activity during the early stages of evolution of a new activity have not been addressed experimentally. We report here an investigation of the evolution of a new activity under circumstances in which both the original and the new activity are critical for growth. Glutamylphosphate reductase (ProA) has a very low promiscuous activity with N-acetylglutamylphosphate, the normal substrate for ArgC (N-acetylglutamylphosphate reductase). A mutation that changes Glu-383 to Ala increases the promiscuous activity by 12-fold but decreases the original activity by 2,800-fold. The impairment in Pro and Arg synthesis results in 14-fold overexpression of E383A ProA, providing sufficient N-acetylglutamylphosphate reductase activity to allow a strain lacking ArgC to grow on glucose. Thus, reaching the threshold level of NAGP reductase activity required for survival required both a structural mutation and overexpression of the enzyme. Notably, overexpression does not require a mutation in the regulatory region of the protein; amino acid limitation attributable to the poor catalytic abilities of E383A ProA causes a physiological response that results in overexpression.

The possibility of alternative microbial life on Earth

Despite its amazing morphological diversity, life as we know it on Earth today is remarkably similar in its basic molecular architecture and biochemistry. The assumption that all life on Earth today shares these molecular and biochemical features is part of the paradigm of modern biology. This paper examines the possibility that this assumption is false, more specifically, that the contemporary Earth contains as yet unrecognized alternative forms of microbial life. The possibility that more than one form of life arose on Earth is consistent with our current understanding of conditions on the early Earth and the biochemical and molecular possibilities for life. Arguments that microbial descendents of an alternative origin of life could not co-exist with familiar life are belied by what we know of the complexity and diversity of microbial communities. Furthermore, the tools that are currently used to explore the microbial world – microscopy (with the aid of techniques such as DAPI staining and fluorescence in situ hybridization), cultivation and PCR amplification of rRNA genes – could not detect such organisms if they existed. Thus, the fact that we have not discovered any alternative life forms cannot be taken as evidence that they do not exist.