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PubMed1982

Overlapping evolutionary affinities revealed by comparison of amino acid compositions.

Comparison of the amino acid compositions of purified proteins indicates the presence of overlapping evolutionary affinities among enzymes of the beta-ketoadipate pathway. Isofunctional enzymes from different bacterial genera share a common evolutionary origin. Moreover, enzymes that mediate isofunctional or chemically analogous reactions within an organism appear to be evolutionarily homologous. Most remarkably, closely similar amino acid compositions are found in enzymes that mediate the following consecutive metabolic steps: lactonization, decarboxylation, hydrolysis, and transfer of a thioester bond.

Adipates↗

PubMed2004

Computational design of a biologically active enzyme.

Rational design of enzymes is a stringent test of our understanding of protein chemistry and has numerous potential applications. Here, we present and experimentally validate the computational design of enzyme activity in proteins of known structure. We have predicted mutations that introduce triose phosphate isomerase activity into ribose-binding protein, a receptor that normally lacks enzyme activity. The resulting designs contain 18 to 22 mutations, exhibit 10(5)- to 10(6)-fold rate enhancements over the uncatalyzed reaction, and are biologically active, in that they support the growth of Escherichia coli under gluconeogenic conditions. The inherent generality of the design method suggests that many enzymes can be designed by this approach.

Algorithms↗

PubMed2003

Single eubacterial origin of eukaryotic sulfide:quinone oxidoreductase, a mitochondrial enzyme conserved from the early evolution of eukaryotes during anoxic and sulfidic times.

Mitochondria occur as aerobic, facultatively anaerobic, and, in the case of hydrogenosomes, strictly anaerobic forms. This physiological diversity of mitochondrial oxygen requirement is paralleled by that of free-living alpha-proteobacteria, the group of eubacteria from which mitochondria arose, many of which are facultative anaerobes. Although ATP synthesis in mitochondria usually involves the oxidation of reduced carbon compounds, many alpha-proteobacteria and some mitochondria are known to use sulfide (H2S) as an electron donor for the respiratory chain and its associated ATP synthesis. In many eubacteria, the oxidation of sulfide involves the enzyme sulfide:quinone oxidoreductase (SQR). Nuclear-encoded homologs of SQR are found in several eukaryotic genomes. Here we show that eukaryotic SQR genes characterized to date can be traced to a single acquisition from a eubacterial donor in the common ancestor of animals and fungi. Yet, SQR is not a well-conserved protein, and our analyses suggest that the SQR gene has furthermore undergone some lateral transfer among prokaryotes during evolution, leaving the precise eubacterial lineage from which eukaryotes obtained their SQR difficult to discern with phylogenetic methods. Newer geochemical data and microfossil evidence indicate that major phases of early eukaryotic diversification occurred during a period of the Earth's history from 1 to 2 billion years before present in which the subsurface ocean waters contained almost no oxygen but contained high concentrations of sulfide, suggesting that the ability to deal with sulfide was essential for prokaryotes and eukaryotes during that time. Notwithstanding poor resolution in deep SQR phylogeny and lack of a specifically alpha-protebacterial branch for the eukaryotic enzyme on the basis of current lineage sampling, a single eubacterial origin of eukaryotic SQR and the evident need of ancient eukaryotes to deal with sulfide, a process today germane to mitochondrial quinone reduction, are compatible with the view that eukaryotic SQR was an acquisition from the mitochondrial endosymbiont.

Amino Acid Sequence↗

PubMed1978

Theoretical considerations of the sensitivity of quantitative subunit hybridization.

The theoretical basis of the quantitative subunit hybridization technique and its ability to measure evolutionary amino acid substitutions is examined. Homospecific:heterospecific enzyme ratios found after subunit reassociation depend upon K1.2, the equilibrium constant for the dissociation of the heterospecific enzyme. It is shown that if this constant is near the geometric mean of the two homospecific enzyme dissociation constants, as it should be in enzymes whose subunits pair isologously, the quantitative subunit hybridization method will not detect most changes in the subunit contact regions of homologous proteins.

Biological Evolution↗

PubMed1992

Evolutionary optimization of the catalytic efficiency of enzymes.

1. The rate equation for a generalized Michaelian type of enzymic reaction mechanism has been analyzed in order to establish how the mechanism should be kinetically designed in order to optimize the catalytic efficiency of the enzyme for a given average magnitude of true and apparent first-order rate constants in the mechanism at given concentrations of enzyme, substrate and product. 2. As long as on-velocity constants for substrate and product binding to the enzyme have not reached the limiting value for a diffusion-controlled association process, the optimal state of enzyme operation will be characterized by forward (true and apparent) first-order rate constants of equal magnitude and reverse rate constants of equal magnitude. The drop in free energy driving the catalysed reaction will occur to an equal extent for each reaction step in the mechanism. All internal equilibrium constants will be of equal magnitude and reflect only the closeness of the catalysed reaction to equilibrium conditions. 3. When magnitudes of on-velocity constants for substrate and product binding have reached their upper limits, the optimal kinetic design of the reaction mechanism becomes more complex and has to be established by numerical methods. Numerical solutions, calculated for triosephosphate isomerase, indicate that this particular enzyme may or may not be considered to exhibit close to maximal efficiency, depending on what value is assigned to the upper limit for a ligand association rate constant. 4. Arguments are presented to show that no useful information on the evolutionary optimization of the catalytic efficiency of enzymes can be obtained by previously taken approaches that are based on the application of linear free-energy relationships for rate and equilibrium constants in the reaction mechanism.

Biological Evolution↗

PubMed2002

The historical biogeography of two Caribbean butterflies (Lepidoptera: Heliconiidae) as inferred from genetic variation at multiple loci.

Mitochondrial DNA and allozyme variation was examined in populations of two Neotropical butterflies, Heliconius charithonia and Dryas iulia. On the mainland, both species showed evidence of considerable gene flow over huge distances. The island populations, however, revealed significant genetic divergence across some, but not all, ocean passages. Despite the phylogenetic relatedness and broadly similar ecologies of these two butterflies, their intraspecific biogeography clearly differed. Phylogenetic analyses of mitochondrial DNA sequences revealed that populations of D. iulia north of St. Vincent are monophyletic and were probably derived from South America. By contrast, the Jamaican subspecies of H. charithonia rendered West Indian H. charithonia polyphyletic with respect to the mainland populations; thus, H. charithonia seems to have colonized the Greater Antilles on at least two separate occasions from Central America. Colonization velocity does not correlate with subsequent levels of gene flow in either species. Even where range expansion seems to have been instantaneous on a geological timescale, significant allele frequency differences at allozyme loci demonstrate that gene flow is severely curtailed across narrow ocean passages. Stochastic extinction, rapid (re)colonization, but low gene flow probably explain why, in the same species, some islands support genetically distinct and nonexpanding populations, while nearby a single lineage is distributed across several islands. Despite the differences, some common biogeographic patterns were evident between these butterflies and other West Indian taxa; such congruence suggests that intraspecific evolution in the West Indies has been somewhat constrained by earth history events, such as changes in sea level.

Animals↗

PubMed1996

Regulation of matrilysin in the rat uterus.

Matrilysin was first discovered in the involuting rat uterus; it has also been known as uterine metalloproteinase, putative metalloproteinase (Pump-1), and matrix metalloproteinase 7 (MMP-7). It is the smallest member (28 kDa) of a family of 15 MMPs that together are able to degrade most of the macromolecules of the extracellular matrix. This family is briefly reviewed; all members are zinc metalloproteinases that occur in zymogen form with the active site zinc blocked by cysteine. Matrilysin can degrade a wide range of gelatins, proteoglycans, and glycoproteins of the matrix and can activate several other MMPs including collagenase. With respect to the uterus, matrilysin is localized to epithelial cells and varies in amount with the estrus cycle and is found in high levels during postpartum involution. There is evidence for a role in the last stage of cervical ripening and immediately postpartum. Induction of premature delivery by onapristone and prostaglandin E2 advances these changes in matrilysin. Regulation of the enzyme levels in the uterus are considered from four viewpoints: control of protein synthesis (particularly in response to hormones), activation of the proenzyme to functional protease, retention of enzyme by binding to matrix components such as heparan sulfate, and inhibition by natural inhibitors such as tissue inhibitor of metalloproteinases (TIMPs) and alpha 2-macroglobulin.

Amino Acid Sequence↗

PubMed2006

Directed evolution of an artificial bifunctional enzyme, gamma-glutamyl kinase/gamma-glutamyl phosphate reductase, for improved osmotic tolerance of Escherichia coli transformants.

To produce the artificial bifunctional enzyme gamma-glutamyl kinase/gamma-glutamyl phosphate reductase, a mutant library of the proBA fusion gene from Bacillus subtilis was created by error-prone PCR. Selecting by functional complementation of the proline auxotroph Escherichia coli JM83 and NaCl tolerance, we isolated a mutant of the proBA fusion gene that improved the osmotolerance of host cells of E. coli JM83. A single amino acid replacement (Asn177Asp) located in a conserved domain in gamma-glutamyl kinase leads to overproduction of proline by host cells. The mutated gamma-glutamyl kinase/gamma-glutamyl phosphate reductase enzyme was rendered about 100-fold less sensitive to proline-mediated feedback inhibition than the control.

Amino Acid Sequence↗

PubMed2005

Synthesis of screening substrates for the directed evolution of sialic acid aldolase: towards tailored enzymes for the preparation of influenza A sialidase inhibitor analogues.

The stereoselective synthesis of two epimeric screening substrates, (4R, 5R, 6R)- and (4S, 5R, 6R)-6-dipropylcarbamoyl-2-oxo-4,5,6-trihydroxy-hexanoic acid, for the directed evolution of sialic acid aldolase is described. The complementary methods relied on stereoselective indium-mediated additions of ethyl alpha-bromomethyl acrylate to functionalised aldehydes. With an alpha-hydroxy aldehyde, (2R, 3R)-2,3-dihydroxy-4-oxo butanoic acid dipropylamide, the addition was chelation controlled, and the syn product, (6R, 5R, 4S)-6-dipropylcarbamoyl-2-methylidene-4,5,6-trihydroxy-hexanoic acid ethyl ester, was obtained. In contrast, the stereochemical outcome of the addition to (2R, 3R)-N,N-dipropyl-2,3-O-isopropylidene-4-oxobutyramide was consistent with Felkin-Anh control, and the anti adduct, (4R, 5R, 6R)-6-dipropylcarbamoyl-2-methylidene-4-hydroxy-5,6-O-isopropylidene-hexanoic acid ethyl ester, was the major product. Ozonolysis and deprotection gave the screening substrates as mixtures of furanose and pyranose forms, in good yields.

Directed Molecular Evolution↗

PubMed2003

Functional fingerprints of folds: evidence for correlated structure-function evolution.

Using structural similarity clustering of protein domains: protein domain universe graph (PDUG), and a hierarchical functional annotation: gene ontology (GO) as two evolutionary lenses, we find that each structural cluster (domain fold) exhibits a distribution of functions that is unique to it. These functional distributions are functional fingerprints that are specific to characteristic structural clusters and vary from cluster to cluster. Furthermore, as structural similarity threshold for domain clustering in the PDUG is relaxed we observe an influx of earlier-diverged domains into clusters. These domains join clusters without destroying the functional fingerprint. These results can be understood in light of a divergent evolution scenario that posits correlated divergence of structural and functional traits in protein domains from one or few progenitors.

Adenosine Triphosphate↗

PubMed2003

Finding evolutionary relations beyond superfamilies: fold-based superfamilies.

Superfamily classifications are based variably on similarity of sequences, global folds, local structures, or functions. We have examined the possibility of defining superfamilies purely from the viewpoint of the global fold/function relationship. For this purpose, we first classified protein domains according to the beta-sheet topology. We then introduced the concept of kinship relations among the classified beta-sheet topology by assuming that the major elementary event leading to creation of a new beta-sheet topology is either an addition or deletion of one beta-strand at the edge of an existing beta-sheet during the molecular evolution. Based on this kinship relation, a network of protein domains was constructed so that the distance between a pair of domains represents the number of evolutionary events that lead one from the other domain. We then mapped on it all known domains with a specific core chemical function (here taken, as an example, that involving ATP or its analogs). Careful analyses revealed that the domains are found distributed on the network as >20 mutually disjointed clusters. The proteins in each cluster are defined to form a fold-based superfamily. The results indicate that >20 ATP-binding protein superfamilies have been invented independently in the process of molecular evolution, and the conservative evolutionary diffusion of global folds and functions is the origin of the relationship between them.

Adenosine Triphosphate↗

PubMed2003

Controlling chirality.

New strategies are continually being developed for using enzymes to efficiently catalyse the enantioselective synthesis of chiral non-racemic compounds. Alternatives to asymmetric synthesis or kinetic resolution include dynamic kinetic resolution, deracemisation and enantioconvergent transformations. Moreover, a much greater understanding is being developed of the parameters that can affect the stereochemical outcome of the reaction (e.g. solvent, substrate design, immobilization and directed evolution).

Catalysis↗

PubMed1988

Interpreting the behavior of enzymes: purpose or pedigree?

To interpret the growing body of data describing the structural, physical, and chemical behaviors of biological macromolecules, some understanding must be developed to relate these behaviors to the evolutionary processes that created them. Behaviors that are the products of natural selection reflect biological function and offer clues to the underlying chemical principles. Nonselected behaviors reflect historical accident and random drift. This review considers experimental data relevant to distinguishing between nonfunctional and functional behaviors in biological macromolecules. In the first segment, tools are developed for building functional and historical models to explain macromolecular behavior. These tools are then used with recent experimental data to develop a general outline of the relationship between structure, behavior, and natural selection in proteins and nucleic acids. In segments published elsewhere, specific functional and historical models for three properties of enzymes--kinetics, stereospecificity, and specificity for cofactor structures--are examined. Functional models appear most suitable for explaining the kinetic behavior of proteins. A mixture of functional and historical models appears necessary to understand the stereospecificity of enzyme reactions. Specificity for cofactor structures appears best understood in light of purely historical models based on a hypothesis of an early form of life exclusively using RNA catalysis.

Binding Sites↗

PubMed1999

Chance favors the prepared genome.

Most descriptions of mutation have emphasized its negative consequences, and randomness with respect to biological function. This book seeks to balance the discussion by emphasizing mechanisms that both diversify the genome and increase the probability that a genome's descendants will survive. This chapter provides a framework for, and overview of, the diverse contributions to this book; these contributions will be stimulating companions, well into the 21st Century, as we work to comprehend the information contained in genomic databases. Genomes that encode "better" amino acid sequences are at a selective advantage. Genomes that generate diversity also are at an advantage to the extent that they can navigate efficiently through the space of possible sequence changes. Biochemical systems that tend to increase the ratio of useful to destructive genetic change may harness preexisting information (horizontal gene transfer, DNA translocation and/or DNA duplication), focus the location, timing, and extent of genetic change, adjust the dynamic range of a gene's activity, and/or sample regulatory connections between sites distributed across the genome. Rejecting entirely random genetic variation as the substrate of genome evolution is not a refutation, but rather provides a deeper understanding, of the theory of natural selection of Darwin and Wallace. The fittest molecular strategies survive, along with descendants of the genomes that encode them.

Animals↗

PubMed2002

Optimizing lipases and related enzymes for efficient application.

Although numerous reactions have been performed using lipases and related enzymes (e.g. esterases and phospholipases), it is still a challenge to identify the most suitable biocatalyst and best reaction conditions for an efficient application. Frequently used methods such as immobilization and optimization of the reaction medium cannot be transferred from one reaction system or substrate to another. However, in the past few years, rational protein design and directed evolution have emerged as efficient alternative methods to optimize biocatalytic reactions.

Bacteria↗

PubMed2001

Kinetic and mechanistic studies of prolyl oligopeptidase from the hyperthermophile Pyrococcus furiosus.

Prolyl oligopeptidase (POP) is widely distributed in mammals, where it is implicated in neuropeptide processing. It is also present in some bacteria and archaea. Because POP is found in mesophilic and hyperthermophilic organisms, and is distributed among all three phylogenetic domains, studies of its function and structure could lead to new insights about the evolution of enzyme mechanisms and thermostability. Kinetic studies were conducted on the POP of the hyperthermophilic archaeon Pyrococcus furiosus (Pfu) 85 degrees C in both H(2)O and D(2)O. Pfu POP displayed many similarities to mammalian POPs, however the solvent isotope effect (k(0)/k(1)) was 2.2 at both high and low pH, indicating that general base/acid catalysis is the rate-limiting step. The pH-rate profiles indicated a three-deprotonation process with pK(a) values of 4.3, 7.2, and 9.1. The temperature dependence of these values revealed a heat of ionization of 4.7 kJ/mol for pK(es1) and 22 kJ/mol for pK(es2), suggesting the catalytic involvement of a carboxyl group and an imidazole group, respectively. Temperature dependence of the catalytic rate was assessed at pH 6.0 and 7.6. Entropy values of -119 and -143 Jmol(-1)K(-1) were calculated at the respective pH values, with a corresponding difference in enthalpy of 8.5 kJ/mol. These values suggest that two or three hydrogen bonds are broken during the transition state of the acidic enzyme form, whereas only one or two are broken during the transition state of the basic enzyme form. A model has been constructed for Pfu POP based on the crystal structure of porcine POP and the sequence alignment. The similarities demonstrated for POPs from these two organisms reflect the most highly conserved characteristics of this class of serine protease, whereas the differences between these enzymes highlights the large evolutionary distance between them. Such fundamental information is crucial to our understanding of the function of proteins at high temperature.

Amino Acid Sequence↗

PubMed2006

Directed evolution and substrate specificity profile of homing endonuclease I-SceI.

The laboratory evolution of enzymes with tailor-made DNA cleavage specificities would represent new tools for manipulating genomes and may enhance our understanding of sequence-specific DNA recognition by nucleases. Below we describe the development and successful application of an efficient in vivo positive and negative selection system that applies evolutionary pressure either to favor the cleavage of a desired target sequence or to disfavor the cleavage of nontarget sequences. We also applied a previously described in vitro selection method to reveal the comprehensive substrate specificity profile of the wild-type I-SceI homing endonuclease. Together these tools were used to successfully evolve mutant I-SceI homing endonucleases with altered DNA cleavage specificities. The most highly evolved enzyme cleaves the target mutant DNA sequence with a selectivity that is comparable to wild-type I-SceI's preference for its cognate substrate.

DNA↗

PubMed2006

Proton-coupled electron transfer: the mechanistic underpinning for radical transport and catalysis in biology.

Charge transport and catalysis in enzymes often rely on amino acid radicals as intermediates. The generation and transport of these radicals are synonymous with proton-coupled electron transfer (PCET), which intrinsically is a quantum mechanical effect as both the electron and proton tunnel. The caveat to PCET is that proton transfer (PT) is fundamentally limited to short distances relative to electron transfer (ET). This predicament is resolved in biology by the evolution of enzymes to control PT and ET coordinates on highly different length scales. In doing so, the enzyme imparts exquisite thermodynamic and kinetic controls over radical transport and radical-based catalysis at cofactor active sites. This discussion will present model systems containing orthogonal ET and PT pathways, thereby allowing the proton and electron tunnelling events to be disentangled. Against this mechanistic backdrop, PCET catalysis of oxygen-oxygen bond activation by mono-oxygenases is captured at biomimetic porphyrin redox platforms. The discussion concludes with the case study of radical-based quantum catalysis in a natural biological enzyme, class I Escherichia coli ribonucleotide reductase. Studies are presented that show the enzyme utilizes both collinear and orthogonal PCET to transport charge from an assembled diiron-tyrosyl radical cofactor to the active site over 35A away via an amino acid radical-hopping pathway spanning two protein subunits.

Amino Acids↗