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A novel scenario for the evolution of haem-copper oxygen reductases.

The increasing sequence information on oxygen reductases of the haem-copper superfamily, together with the available three-dimensional structures, allows a clear identification of their common, functionally important features. Taking into consideration both the overall amino acid sequences of the core subunits and key residues involved in proton transfer, a novel hypothesis for the molecular evolution of these enzymes is proposed. Three main families of oxygen reductases are identified on the basis of common features of the core subunits, constituting three lines of evolution: (i) type A (mitochondrial-like oxidases), (ii) type B (ba3-like oxidases) and (iii) type C (cbb3-type oxidases). The first group can be further divided into two subfamilies, according to the helix VI residues at the hydrophobic end of one of the proton pathways (the so-called D-channel): (i) type A1, comprising the enzymes with a glutamate residue in the motif -XGHPEV-, and (ii) type A2, enzymes having instead a tyrosine and a serine in the alternative motif -YSHPXV-. This second subfamily of oxidases is shown to be ancestor to the one containing the glutamate residue, which in the Bacteria domain is only present in oxidases from Gram-positive or purple bacteria. It is further proposed that the Archaea domain acquired terminal oxidases by gene transfer from the Gram-positive bacteria, implying that these enzymes were not present in the last common ancestor before the divergence between Archaea and Bacteria. In fact, most oxidases from archaea have a higher amino acid sequence identity and similarity with those from bacteria, mainly from the Gram-positive group, than with oxidases from other archaea. Finally, a possible relation between the dihaemic subunit (FixP) of the cbb3 oxidases and subunit II of caa3 oxidases is discussed. As the families of haem-copper oxidases can also be identified by their subunit II, a parallel evolution of subunits I and II is suggested.

Amino Acid Sequence↗

Ancestral lysozymes reconstructed, neutrality tested, and thermostability linked to hydrocarbon packing.

The controversy surrounding the idea that neutral mutations dominate protein evolution is attributable in part to the inadequacy of the tools available to evolutionary investigators. With a few exceptions, most investigations into the force driving protein evolution have relied on indirect criteria for distinguishing neutral and non-neutral variants. To investigate a particular pathway of molecular evolution, we have reconstructed by site-directed mutagenesis likely ancestral variants of the lysozymes of modern game birds (order Galliformes), tested their activity and thermostability and determined their three-dimensional structure. We focused on amino acids at three positions that are occupied in all known game birds either by the triplet Thr 40, Ile 55, Ser 91, or by the triplet Ser 40, Val 55, Thr 91. We have synthesized proteins representing intermediates along the possible three-step evolutionary pathways between these triplets. Although all of these are active and stable, none of these intermediates is found in known lysozymes. A comparison of the structures and thermostabilities of the variants reveals a linear correlation between the side-chain volume of the triplet and the thermostability of the protein. Each pathway connecting the two extant triplet sequences includes a variant with a thermostability outside the range of the extant proteins. This observation is consistent with a non-neutral evolutionary pathway. The existence of variants that are more stable than the extant proteins suggests that selection for maximum thermostability may not have been an important factor in the evolution of this enzyme.

Amino Acid Sequence↗

Time-dependent closed form solutions for fully competitive enzyme reactions.

An analytic formalism developed earlier to describe the time evolution of the basic enzyme reaction is extended to fully competitive systems. Time-dependent closed form solutions are derived for the three nominal cases of competition: even, slow and fast inhibitors, allowing for the first time the complete characterization of the reactions. In agreement with previous work, the time-independent Michaelis-Menten approach is shown to be inaccurate when a fast inhibitor is present. The validity of the quasi-steady-state approximation on which the present framework is based is also revised.

Enzyme Inhibitors↗

The (betaalpha)(8) glycosidases: sequence and structure analyses suggest distant evolutionary relationships.

There are currently at least nine distinct glycosidase sequence families which are all known to adopt a TIM barrel fold [Henrissat,B. and Davies,G. (1997) CURR: Opin. Struct. Biol., 7, 637-644]. To explore the relationships between these enzymes and their evolution, comprehensive sequence and structure comparisons were performed, generating four distinct clusters. The first cluster, S1, comprises the alpha-amylase related enzymes, all with the retention mechanism (axial-->axial). The second cluster, S2, included two functional subgroups, one composed of various kinds of glucosidases all with the retention mechanism (equatorial-->equatorial) (the so-called 4/7 superfamily), and the other subgroup including the beta-amylases with the inversion mechanism (axial--> equatorial). The third cluster, S3, with the retention mechanism (equatorial-->equatorial), could be subdivided, based on the catalytic residues and mechanisms, into two functional subgroups: the chitinase group, catalysed by two acidic residues on the C-termini of beta-4 and beta-6, and the hevamine group, using two acidic residues on the C-termini of beta-4 for catalysis. The fourth cluster, S4, is composed of chitobiase with the retention mechanism (equatorial--> equatorial). These clusters are compared with the sequence families derived by Henrissat and coworkers. PSI-BLAST profiles and multiple-alignments of tertiary structures suggest that S1 and S2 are distantly related, as are S3 and S4, which have N-acetylated substrates. This work highlights the difficulties of untangling distant evolutionary relationships in ubiquitous folds such as the TIM barrel.

Algorithms↗

Modelling the three-dimensional structure and electrostatic potential field of the two Cu,Zn superoxide dismutase variants from Xenopus laevis.

The crystallographic structure of bovine superoxide dismutase has been used as a template for the graphic reconstruction of the three-dimensional structures of the two Xenopus laevis variants (Schininà, M.E. et al. Arch. Biochem. Biophys. 272:507-515, 1989). In these models the structure-essential residues maintain their position and their structural role, and the interactions between the subunits and the close packing within the beta-barrel are maintained with conservative substitutions and even increased with "aromatic pairs." Because of the same topological motif and surface location of charges, arising from the model building of the two variants with respect to the bovine enzyme, we have calculated the electrostatic potential fields around the models of the two Xenopus laevis variants by numerically solving the Poisson-Boltzmann equation. We show that conservation of a specific space-relationship of charges maintains the potential field pattern already observed in the bovine enzyme, where a negative potential field surrounds the protein surface and specific positive regions wrap up the copper center active site. This electrostatic potential field distribution supports the idea that electrostatic interactions control, like in the bovine enzyme, the mechanism of enzyme-substrate recognition in the Xenopus laevis Cu,Zn superoxide dismutases, suggesting that coordinated mutation of charged residues has occurred in the evolution of this enzyme.

Amino Acid Sequence↗

Sexually dimorphic expression of glutamate decarboxylase mRNA in the hypothalamus of the deep sea armed grenadier, Coryphaenoides (Nematonurus) armatus.

Glutamate decarboxylase (GAD), is a key enzyme in the central nervous system (CNS) that synthesizes the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) from glutamate. Our previous phylogenetic studies on the evolution of this enzyme indicates that there are at least two distinct forms: GAD65 and GAD67. They are the products of separate genes and probably derive from a common ancestral GAD gene following gene duplication prior to the emergence of the teleosts more than 200 Myr ago. Furthermore, a third GAD-like molecule, GAD3, discovered in the armed grenadier, Coryphaenoides (Nematonurus) armatus, is equally divergent from both GAD65 and GAD67. Specimens of C. (N.) armatus were collected by trawl at a depth of 4,000 m in the Porcupine Seabight (Northeastern Atlantic), and brains dissected and frozen for RNA extraction. All three GAD forms are found in the cerebellum, telencephalon and hypothalamus. Semiquantitative PCR analysis showed that males and females have similar levels of expression of GAD67 and GAD3 in the tissues studied. Independent of the sex examined, the levels of expression of GAD65 and GAD67 in the cerebellum were approximately half that in the telencephalon. GAD3 levels were approximately 30% higher in the cerebellum than in either the telencephalon or hypothalamus. In contrast to GAD67 and GAD3, hypothalamic expression of GAD65 mRNA is 1.8 times higher (p < 0.05) in males than in females. These data indicate that the expression of GAD65, a key enzyme for the synthesis of GABA is sexually dimorphic in females and males of C. (N.) armatus.

Animals↗

Modelling, steady state analysis and optimization of the catalytic efficiency of the triosephosphate isomerase.

In the present work we have modelled and optimized the reaction mechanism of the triose phosphate isomerase (TIM) enzyme (E.C. 5.3.1.1). For this purpose we have used an approach that combines the S-system representation within the power law formalism and linear programming techniques. By this means we have explored those rate constants whose alterations are likely to improve the catalytic efficiency of the enzyme and investigated the available room for optimization in different metabolic conditions. The role and plausibility of the different types of mutations on the evolution of this enzyme have also been considered. Steady state sensitivity analysis was carried out and a new set of aggregated logarithmic gains was defined in order to quantify the responses of the system to changes in groups of rate constants that could be explained in terms of mutations affecting the catalytic properties of the enzyme. Evaluation of these logarithmic gains at different levels of saturation and disequilibrium ratios enabled us to reach conclusions about the meaning and role of the diffusion limitation terms. The catalytic efficiency of the monoenzymatic system was optimized through changes in the kinetic rate constants within different sets of restrictions ranging from thermodynamic or kinetic to evolutionary ones. Results showed that, at very different conditions, there is still room for improvement in the TIM enzyme. Thus, in a wide range of metabolically significant values of the disequilibrium ratio there is a minimal variation in the optimal profile that yields 2.1 times the velocity of the basal states. Though most of this increase is accounted for by the increase of the second order constants (that could have already reached a theoretical maximum) significant increases (20%) in catalytic efficiencies are obtained by changes of the internal steps only. Besides these new findings our optimization approach has been able to reproduce results obtained with other approaches.

Catalysis↗

Small peptides as modular catalysts for the direct asymmetric aldol reaction: ancient peptides with aldolase enzyme activity.

Simple peptides and their analogues having a primary amino group as the catalytic residue mediate the direct asymmetric intermolecular aldol reaction with high stereoselectivity and furnish the corresponding aldol products with up to 99% ee; this intrinsic ability of highly modular peptides may explain the initial molecular evolution of aldolase enzymes.

Catalysis↗

Glutathione transferases--structure and catalytic activity.

The glutathione transferases are recognized as important catalysts in the biotransformation of xenobiotics, including drugs as well as environmental pollutants. Multiple forms exist, and numerous transferases from mammalian tissues, insects, and plants have been isolated and characterized. Enzymatic properties, reactions with antibodies, and structural characteristics have been used for classification of the glutathione transferases. The cytosolic mammalian enzymes could be grouped into three distinct classes--Alpha, Mu, and Pi; the microsomal glutathione transferase differs greatly from all the cytosolic enzymes. Members of each enzyme class have been identified in human, rat, and mouse tissues. Comparison of known primary structures of representatives of each class suggests a divergent evolution of the enzyme proteins from a common precursor. Products of oxidative metabolism such as organic hydroperoxides, epoxides, quinones, and activated alkenes are possible "natural" substrates for the glutathione transferases. Particularly noteworthy are 4-hydroxyalkenals, which are among the best substrates found. Homologous series of substrates give information about the properties of the corresponding binding site. The catalytic mechanism and the active-site topology have been probed also by use of chiral substrates. Steady-state kinetics have provided evidence for a "sequential" mechanism.

Catalysis↗

Adaptation of Drosophila enzymes to temperature. III. Evolutionary conservation in mitochondrial enzymes.

The evolutionary behavior of two mitochondrial enzymnes (L-glycerol 3-phosphate:cytochrome c oxidoreductase E.C.1.1.1.95, alpha GPO, and L-malate: NAD+ oxidoreductase, E.C.1.1.1.37, m-MDH) obtained from several temperate and tropical Drosophila species was examined by comparing their catalytic properties, which related to temperature (Km-Ea-Q10-Thermostability). Mitochondrial alpha GPO or m-MDH obtained either from template or from tropical species was found to exhibit similar catalytic properties while for both cytosolic enzymes, the alpha GPDH and s-MDH, Km patterns were similar among species from the same thermal habitat and different thermal habitats. In combination with other observations reported in the literature these facts support the view that the function, and probably the structure, of mitochondrial enzymes are better conserved in evolution than those of the corresponding enzymes found in the cytosol. It is proposed that the relative invariance of the mitochondrial enzymes structure is probably linked to a necessary relative invariance of molecular interactions inside the mitochondrion.

Animals↗

Dynamics of cellulase production by glucose grown cultures of Trichoderma reesei Rut-C30 as a response to addition of cellulose.

An economic process for the enzymatic hydrolysis of cellulose would allow utilization of cellulosic biomass for the production of easily fermentable low-cost sugars. New and more efficient fermentation processes are emerging to convert this biologic currency to a variety of commodity products with a special emphasis on fuel ethanol production. Since the cost of cellulase production currently accounts for a large fraction of the estimated total production costs of bioethanol, a significantly less expensive process for cellulase enzyme production is needed. It will most likely be desirable to obtain cellulase production on different carbon sources-including both polymeric carbohydrates and monosaccharides. The relation between enzyme production and growth profile of the microorganism is key for designing such processes. We conducted a careful characterization of growth and cellulase production by the soft-rot fungus Trichoderma reesei. Glucose-grown cultures of T. reesei Rut-C30 were subjected to pulse additions of Solka-floc (delignified pine pulp), and the response was monitored in terms of CO2 evolution and increased enzyme activity. There was an immediate and unexpectedly strong CO2 evolution at the point of Solka-floc addition. The time profiles of induction of cellulase activity, cellulose degradation, and CO2 evolution are analyzed and discussed herein.

Biomass↗

Phylogenetic survey of endomannosidase indicates late evolutionary appearance of this N-linked oligosaccharide processing enzyme.

Endo-alpha-D-mannosidase is a processing enzyme which in contrast to other glycosidases involved in the trimming of N-linked oligosaccharides of glycoproteins acts at an internal position by cleaving the linkage between the glucose-substituted mannose and the internal portion of the polymannose unit and thereby provides an alternate deglucosylating pathway. In order to evaluate at what stage in evolution this unusual enzyme first emerged, we have carried out a phylogenetic survey of its distribution among a broad group of eukaryotes ranging from unicellular organisms to highly developed animals and plants, all of which are known to have the capacity to N-glycosylate proteins and subsequently trim the nascent glucosylated polymannose oligosaccharides. It became evident from enzyme assays and in vivo studies that endomannosidase is limited in its distribution to members of the chordate phylum, including placental and marsupial mammals, birds, reptiles, amphibians, and fish, with the single except of the Mollusca in which it was detected in three distinct classes. The enzyme's absence in all other invertebrates examined as well as in yeast, various protozoa and higher plants, stands in contrast to glucosidase II and alpha 1,2-mannosidase which were found to be present in all eukaryotes studied. The observation that endomannosidase activity was not present in insects was confirmed by radiolabeling experiments with Sf9 cells in culture. These cells, which are widely employed for the expression of mammalian genes, were in distinction to mouse cells unable to circumvent a castanospermine (CST)-induced glucosidase blockade. Moreover we observed that Tetrahymenae, which synthesize glycoproteins with truncated N-linked oligosaccharides, could not process these beyond the Glc3Man5GlcNAc2 stage in the presence of CST. The late appearance of endomannosidase during evolution suggests a need for an alternate deglucosylation route in higher animals which parallels the development of elaborate complex N-linked oligosaccharides. Such carbohydrate units are believed to carry out vital biological functions and deglucosylation is a prerequisite to the further processing steps required for their formation.

Animals↗

Glutamate synthase is plastid-encoded in a red alga: implications for the evolution of glutamate synthases.

An actively transcribed gene (glsF) encoding for ferredoxin-dependent glutamate synthase (Fd-GOGAT) was found on the plastid genome of the multicellular red alga Antithamnion sp. Fd-GOGAT is not plastid-encoded in chlorophytic plants, demonstrating that red algal plastid genomes encode for additional functions when compared to those known from green chloroplasts. Moreover, our results suggest that the plant Fd-GOGAT has an endosymbiotic origin. The same may not be true for NADPH-dependent GOGAT. In Antithamnion glsF is flanked upstream by cpcBA and downstream by psaC and is transcribed monocistronically. Implications of these results for the evolution of GOGAT enzymes and the plastid genome are discussed.

Amino Acid Sequence↗

Nomenclature and functions of RNA-directed RNA polymerases.

There is little relationship between eukaryotic RNA-directed RNA polymerases (RDRs), viral RNA-dependent RNA polymerases (RdRps) and DNA-dependent RNA polymerases, indicating that RDRs evolved as an independent class of enzymes early in evolution. In fungi, plants and several animal systems, RDRs play a key role in RNA-mediated gene silencing [post-transcriptional gene silencing (PTGS) in plants and RNA interference (RNAi) in non-plants] and are indispensable for heterochromatin formation, at least, in Schizosaccharomyces pombe and plants. Recent findings indicate that PTGS, RNAi and heterochromatin formation not only function as host defence mechanisms against invading nucleic acids but are also involved in natural gene regulation. RDRs are required for these processes, initiating a broad interest in this enzyme class.

Evolution, Molecular↗

Codification and evolution of experimentally observed specific recognition sites for restriction enzymes on DNA.

The list of published restriction endonucleases along with their substrates provides an excellent data base for the evaluation of the evolution and codification of the key elements for specific recognition sites on the DNA. In this paper the considerations will be limited to palindromic tetramer-, pentamer-, and hexamer-sequences. It is basically assumed that each base pair within these sequences has to be recognized by directionally unique bidentate hydrogen bonds either within the plane of the base pair or by bridging the appropriate H-bond donor/acceptor groups of the neighbouring bases of the same strand. Thus sequence specificity is mediated by twelve (eight) H-bonds, originating from the protein recognition modules. Besides a pronounced preference for GC base pairs expressed by their high frequency in the most abundant sequences, serving the need of maximal thermodynamic stability of the double helical substrates, it can also be shown that the stacking of consecutive bases within the recognition site sequences plays a major role in shaping the particular DNA/protein interface. Finally it will be demonstrated that the full set of sequences discussed in this paper can readily be derived by stepwise expanding the vocabulary of three simple tetrameric sequences by inserting single base pairs into the centre of a minimal sequence, thus creating all the published pentameric restriction sites, or by inserting/adding two GC base pairs in a palindromic way, thus creating the known multiplicity of hexameric sites.

Base Sequence↗

Enhanced accumulation and changed monomer composition in polyhydroxyalkanoate (PHA) copolyester by in vitro evolution of Aeromonas caviae PHA synthase.

By in vitro evolution experiment, we have first succeeded in acquiring higher active mutants of a synthase that is a key enzyme essential for bacterial synthesis of biodegradable polyester, polyhydroxyalkanoate (PHA). Aeromonas caviae FA440 synthase, termed PhaC(Ac), was chosen as a good target for evolution, since it can synthesize a PHA random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] that is a tough and flexible material compared to polyhydroxybutyrate (PHB) homopolyester. The in vitro enzyme evolution system consists of PCR-mediated random mutagenesis targeted to a limited region of the phaC(Ac) gene and screening mutant enzymes with higher activities based on two types of polyester accumulation system by using Escherichia coli for the synthesis of PHB (by JM109 strain) (S. Taguchi, A. Maehara, K. Takase, M. Nakahara, H. Nakamura, and Y. Doi, FEMS Microbiol. Lett. 198:65-71, 2001) and of P(3HB-co-3HHx) [by LS5218 [fadR601 atoC(Con)] strain]. The expression vector for the phaC(Ac) gene, together with monomer-supplying enzyme genes, was designed to synthesize PHB homopolyester from glucose and P(3HB-co-3HHx) copolyester from dodecanoate. Two evolved mutant enzymes, termed E2-50 and T3-11, screened through the evolution system exhibited 56 and 21% increases in activity toward 3HB-coenzyme A, respectively, and consequently led to enhanced accumulation (up to 6.5-fold content) of P(3HB-co-3HHx) in the recombinant LS5218 strains. Two single mutations in the mutants, N149S for E2-50 and D171G for T3-11, occurred at positions that are not highly conserved among the PHA synthase family. It should be noted that increases in the 3HHx fraction (up to 16 to 18 mol%) were observed for both mutants compared to the wild type (10 mol%).

Acyltransferases↗

Unusual evolution of 11beta- and 17beta-hydroxysteroid and retinol dehydrogenases.

11beta-hydroxysteroid dehydrogenases regulate glucocorticoid concentrations and 17beta-hydroxysteroid dehydrogenases regulate estrogen and androgen concentrations in mammals. Phylogenetic analysis of the sequences from two 11beta-hydroxysteroid dehydrogenases and four mammalian 17beta-hydroxysteroid dehydrogenases indicates unusual evolution in these enzymes. Type 1 11beta- and 17beta-hydroxysteroid dehydrogenases are on the same branch; Type 2 enzymes cluster on another branch with beta-hydroxybutyrate dehydrogenase,11-cis-retinol dehydrogenase and retinol dehydrogenase; Type 3 17beta-hydroxysteroid dehydrogenase is on a third branch; while the pig dehydrogenase clusters with a yeast multifunctional enzyme on a fourth branch. Pig 17beta-hydroxysteroid dehydrogenase appears to have evolved independently from the other three 17beta-hydroxysteroid dehydrogenases; in which case, the evolution of 17beta-hydroxysteroid dehydrogenase activity is an example of functional convergence. The phylogeny also suggests that independent evolution of specificity toward C11 substituents on glucocorticoids and C17 substituents on androgens and estrogens has occurred in Types 1 and 2 11beta- and 17beta-hydroxysteroid dehydrogenases.

11-beta-Hydroxysteroid Dehydrogenases↗

Reconstruction of an enzyme by domain substitution effectively switches substrate specificity.

The polar domains of the two transcarbamoylases, aspartate transcarbamoylase (ATCase) and ornithine transcarbamoylase, (OTCase) from Escherichia coli bind the common substrate carbamoyl phosphate and share extensive amino-acid sequence homology. The equatorial domains of the two enzymes differ in their substrate specificity (ATCase binds aspartate, OTCase binds ornithine) and have decreased sequence identity. While addressing the conservation of specific protein interactions during the evolution of these enzymes, we were able to switch one of their amino-acid-specific equatorial domains to produce a viable chimaeric enzyme. This was achieved by the in vitro fusion of DNA encoding the polar domain of OTCase to DNA encoding the equatorial domain of ATCase. The resulting gene fusion successfully transformed an argI-pyrB deletion strain of E. coli to pyrimidine prototrophy, giving rise to Pyr+ transformants that expressed ATCase but not OTCase activity. The formation of this active chimaeric enzyme shows that by exchanging protein domains between two functionally divergent enzymes we have achieved a switching in substrate specificity.

Aspartate Carbamoyltransferase↗