Structure-based characterization of canine-human chimeric uricases and its evolutionary implications

Uricase was lost in hominoids during primate evolution, but the inactivation mechanism remains controversial. To investigate the inactivation process of hominoid uricase, chimeric constructions between canine and human uricase were employed to screen the target regions that may contain labile or inactivated mutations in deduced human uricase. Four chimeric uricases were constructed and showed different enzymatic characteristics. Homology modeling, rational site-directed mutagenesis and DNA alignment were used to analyze the changes. Arg119 is conserved in functional mammalian uricases and its side-chains are crucial in maintaining the stability of the β-barrel core. A single CGT (Arg) to CAT (His) mutation at codon 119 that is shared by the human and great ape clade greatly reduces this stability and could cause the loss of uricase activity. We speculate that this missense mutation occurred first and inactivated the uricase protein in humans and great apes and that later the known nonsense mutation at codon 33 occurred and silenced the uricase gene. A single GTC (Val) to GCC (Ala) mutation at codon 296 in canine uricase is regarded as deleterious structural mutation, but such kinds of deleterious mutations have been widely accumulated in extant mammalian uricases. We speculate that a reduction in uricase activity has been an evolutionary tendency in mammals. Moreover, from structure-activity analysis of helix 2 in ancestral primate uricase, we suggest that before the inactivation of hominoid uricase, deleterious structural evolutionary changes had occurred in ancestral primates. The loss of hominoid uricase should be caused by progressive multistep mutations rather than a single mutation event.     

Comparison of intraperoxisomal localization form and properties of amphibian (Rana catesbeiana) uricase with those of other animal uricases

Liver uricase of bull frog (Rana catesbeiana) was present as the soluble form in the peroxisomal matrix and consisted of four identical subunits with a molecular weight of 30,000. These properties were identical with those of fish liver uricase but differed from mammalian liver uricase. Purified uricase from the frog liver was insoluble in hypertonic, hypotonic and detergent solutions at pH 6-9. This insolubility was the same as mammalian liver uricase but differed from fish liver uricase; fish uricase was soluble in these solutions. The frog liver uricase did not cross-react immunologically with both uricases of fish and mammalian liver. An immunological cross-reactivity of liver uricase was observed among amphibia.     

Phylogenetic Articulation of Uric Acid Evolution in Mammals and How It Informs a Therapeutic Uricase

The role of uric acid during primate evolution has remained elusive ever since it was discovered over 100 years ago that humans have unusually high levels of the small molecule in our serum. It has been difficult to generate a neutral or adaptive explanation in part because the uricase enzyme evolved to become a pseudogene in apes thus masking typical signals of sequence evolution. Adding to the difficulty is a lack of clarity on the functional role of uric acid in apes. One popular hypothesis proposes that uric acid is a potent antioxidant that increased in concentration to compensate for the lack of vitamin C synthesis in primate species ∼65 Ma. Here, we have expanded on our previous work with resurrected ancient uricase proteins to better resolve the reshaping of uricase enzymatic activity prior to ape evolution. Our results suggest that the pivotal death-knell to uricase activity occurred between 20 and 30 Ma despite small sequential modifications to its catalytic efficiency for the tens of millions of years since primates lost their ability to synthesize vitamin C, and thus the two appear uncorrelated. We also use this opportunity to demonstrate how molecular evolution can contribute to biomedicine by presenting ancient uricases to human immune cells that assay for innate reactivity against foreign antigens. A highly stable and highly catalytic ancient uricase is shown to elicit a lower immune response in more human haplotypes than other uricases currently in therapeutic development.     

Cloning and sequence analysis of cDNA for rat liver uricase

We have isolated cDNA clones for rat liver uricase using an oligonucleotide corresponding to the N-terminal sequence of 8 amino acids. The nucleotide sequences of the cDNAs have been determined, and the amino acid sequence of the protein deduced. A 867-base open reading frame coding for 289 amino acids, corresponding to a molecular mass of 33,274 daltons, was confirmed by matching eight sequences of a total of 53 amino acids from peptide sequence analyses of the fragments generated by lysyl endopeptidase digestion of purified rat liver uricase. The deduced amino acid sequence of rat liver uricase shares 40% homology with that of soybean nodulin-specific uricase and has an N-terminal extension of 7 amino acids. In contrast, soybean uricase has a C-terminal extension of 12 amino acids, which is presumably the result of local gene duplication. Completely different N- and C-terminal structures of the two uricases suggest that the signals for targeting the proteins to the peroxisome are not located on the terminal continuous stretches of amino acids.     

Cloning of rabbit uricase cDNA reveals a conserved carboxy-terminal tripeptide in three species

cDNA clones encoding uricase have been isolated from a rabbit liver cDNA library. The nucleotide sequences of the cDNAs have been determined and those of the rat uricase cDNA have been revised. In all three uricases, the carboxy-terminal tripeptides are Ser-Arg/Lys-Leu sequences, which have recently been suggested as an essential element of peroxisomal targetting signals for many but not all peroxisomal proteins.     

Identification of an amino acid residue involved in the substrate-binding site of rat liver uricase by site-directed mutagenesis.

Computer analysis has shown that a conserved amino acid sequence (Leu 160 to Lys 164) of rat liver uricase is also present in other enzymes with purine substrates. The significances of the amino acids in this sequence were studied by site-directed mutagenesis. Replacement of Lys 164 by Glu or Ile resulted in loss of uricase activity and decrease in binding of the competitive inhibitor xanthine. The far ultraviolet circular dichroic spectra of the mutant uricases were identical to that of the wild type protein, indicating that the replacement of Lys 164 by other amino acids did not result in serious modification of the conformation of uricase. These findings suggest that this amino acid is involved in the substrate-binding site of the enzyme.     

Modified colorimetric assay for uricase activity and a screen for mutant Bacillus subtilis uricase genes following StEP mutagenesis.

This study describes a modified colorimetric assay for uricase activity in flexible 96-well microtiter plates using the uricase/uric acid/horseradish peroxidase/4-aminoantipyrine/3,5-dichloro-2-hydroxybenzene sulfonate colorimetric reaction. The utility of this assay was demonstrated in a screen for mutant uricase enzymes derived from the uricase gene of the thermophilic bacterium Bacillus subtilis by a modified staggered extension process (StEP) mutagenesis. An Escherichia coli library of StEP-derived uricase mutant clones was screened yielding two identical active mutant uricase genes. Two motifs conserved in eukaryotic and prokaryotic uricases are highly conserved in the mutant uricase. The mutant uricase protein was found to exhibit high uricase activity (13.1 U.mg(-1)). Finally, the modified colorimetric method is much more efficient than the conventional ones and greatly reduces assay time from 4 days to less than 20 h.     

Further studies on D-3-aminoisobutyrate-pyruvate aminotransferase

D-3-Aminoisobutyrate-pyruvate aminotransferase (EC 2.6.1.40, D-BAIB aminotransferase) participates in the metabolism of thymine. Recently we purified this enzyme from rat liver. We have studied D-BAIB aminotransferase further to clarify its physiological function. Among our findings were the following. (1) The enzyme activity was widely distributed in the organs of guinea pigs and rats. The kidney, liver, and lung showed high specific activities. (2) Using the livers of six vertebrates, differences between species were studied. Activity was detected in all species, the human liver showing the lowest activity among them. (3) Developmental study using rat liver showed that the activity was low at birth, increased sharply thereafter for 10 days, and then subsequently declined to the adult level. (4) Intraperitoneal injection of BAIB and beta-alanine in rats was performed to determine whether they induce activity of this aminotransferase. Only BAIB increased the activity of the aminotransferase in the liver significantly. (5) Subcellular distribution study of this aminotransferase in rat liver revealed that it is a mitochondrial enzyme.     

Cloning and expression in Escherichia coli of the gene encoding Aspergillus flavus urate oxidase.

Amino acid sequencing of peptides obtained after proteolytic hydrolysis of Aspergillus flavus urate oxidase (uricase) permitted the design of oligodeoxynucleotide probes that were used to obtain 1.2- and 5-kilobase pair DNA fragments from A. flavus cDNA and genomic libraries, respectively. The cDNA fragment contained the entire coding region for uricase, and comparison with the genomic fragment revealed the presence of two short introns in the coding region of the gene. A. flavus uricase has around 40% overall identity with uricases from higher organisms but with many conserved amino acids. Hitherto highly conserved consensus patterns found in other uricases were found to be modified in the A. flavus enzyme, notably the sequence Val-Leu-Lys-Thr-Thr-Gln-Ser near position 150, which in the filamentous fungus is uniquely modified to Val-Leu-Lys-Ser-Thr-Asn-Ser. Silent mutations were introduced by cassette mutagenesis near the 5'-extremity of the coding sequence in order to conform with Escherichia coli codon usage, and the uricase was expressed in the E. coli cytoplasm in a completely soluble, biologically active form.     

Uricase from leaves: its purification and characterization from three different higher plants

Uricase (urate: oxygen oxidoreductase, EC␣1.7.3.3) from leaves of chickpea (Cicer arietimum L.), broad bean (Vicia faba major L.), and wheat (Triticum aestivum L.) has been purified to electrophoretic homogeneity by a procedure which includes xanthine-agarose affinity chromatography as the main step. Purification factors of 74 000–83 000 and recoveries of 80–90% were achieved. Purified preparations had specific activities between 600 and 800 nkat · mg protein⁻¹ (turnover numbers between 4400 and 6400 min⁻¹). The three plant uricases were found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be tetramers of similar molecular mass (120–130 kDa) and to have identical or similar-sized subunits (32–34 kDa). They also had a similar optimum pH (9–9.5) and showed a hyperbolic kinetics with K _m values from 9–24 μM. All of them showed similar responses to putative activators/inhibitors. Oxonate, xanthine and, to a lesser extent, neocuproin inhibited uricase activity, whereas allantoin, ammonium, citrulline and glutamine did not. The three leaf uricases lacked catalase activity and were not activated by cadaverine. None of the three plant enzymes cross-reacted with anti-uricase monoclonal antibodies from soybean nodules or anti-uricase polyclonal antibodies from Chlamydomonas reinhardtii or rat liver. These results are consistent with the view that uricase in plants is probably a unique enzyme which is expressed at very low level in leaves. Received: 28 October 1996 / Accepted: 8 January 1997     

Transposable elements in medaka fish

DNA-based transposable elements appear to have been nearly or completely inactivated in vertebrates. Therefore the elements of the medaka fish Oryzias latipes that still have transposition activity provide precious materials for studying transposition mechanisms, as well as the evolution, of transposable elements in vertebrates. Fortunately, the medaka fish has a strong background for genetic and evolutionary studies. The advantages of this host species and their elements, together with results so far obtained, are here described.     

Isolation and characterization of uricase from bean leaves and its comparison with uredospore enzymes

Uricase (urate: oxygen oxidoreductase; EC 1.7.3.3) from bean (Phaseolus vulgaris) leaves and uredospores of two different rust fungi (Uromyces phaseoli and Uromyces fabae) has been purified to electrophoretic homogeneity by a procedure which includes xanthine–agarose affinity chromatography as the main step. Pure preparations had similar specific activities (2–6 U mg⁻¹) with turnover numbers from 250 to 750 min⁻¹, and all enzymes were tetramers consisting of identical or similar-sized subunits of 32–33 kDa. They also exhibited similar optimum pH (around 9.0), showed hyperbolic kinetics with K_m values of 15–34 μM and behaved similarly against a number of putative activators/inhibitors, all of them being inhibited only by oxonate and xanthine. However, leaf and uredospore uricases differed in the strength of binding to DEAE-cellulose since leaf uricase did not bind to the exchanger and that from U. fabae bound stronger than that of U. phaseoli. Uredospore uricases showed complete antigenic independence against anti-uricase polyclonal antibodies from bean leaves and anti-uricase monoclonal antibodies from soybean nodules. Cross-reaction was observed between leaf uricase and nodule monoclonal antibodies and between nodule enzyme and leaf polyclonal antibodies. These results confirm the homogeneity of plant uricases and demonstrate that fungal obligate parasites have their own uricase, which is similar to the plant enzyme in many molecular and kinetic properties but different in DEAE-cellulose binding characteristics and immunological properties.     

Evolution of behavior and neural control of the fast-start escape response

The fast-start startle behavior is the primary mechanism of rapid escape in fishes and is a model system for examining neural circuit design and musculoskeletal function. To develop a dataset for evolutionary analysis of the startle response, the kinematics and muscle activity patterns of the fast-start were analyzed for four fish species at key branches in the phylogeny of vertebrates. Three of these species (Polypterus palmas, Lepisosteus osseus, and Amia calva) represent the base of the actinopterygian radiation. A fourth species (Oncorhynchus mykiss) provided data for a species in the central region of the teleost phylogeny. Using these data, we explored the evolution of this behavior within the phylogeny of vertebrates. To test the hypothesis that startle features are evolutionarily conservative, the variability of motor patterns and kinematics in fast-starts was described. Results show that the evolution of the startle behavior in fishes, and more broadly among vertebrates, is not conservative. The fast-start has undergone substantial change in suites of kinematics and electromyogram features, including the presence of either a one- or a two-stage kinematic response and change in the extent of bilateral muscle activity. Comparative methods were used to test the evolutionary hypothesis that changes in motor control are correlated with key differences in the kinematics and behavior of the fast-start. Significant evolutionary correlations were found between several motor pattern and behavioral characters. These results suggest that the startle neural circuit itself is not conservative. By tracing the evolution of motor pattern and kinematics on a phylogeny, it is shown that major changes in the neural circuit of the startle behavior occur at several levels in the phylogeny of vertebrates.     

A human genomic sequence highly homologous to the 3'-untranslated region of rat uricase mRNA

A human genomic sequence was isolated from a library using a rat uricase cDNA probe. Sequence analysis has shown that it is highly homologous to the 3'-untranslated region of rat uricase mRNA. Total loss of uricase activity in human is, therefore, not due to total loss of the gene. Discovery of high degree of conservation of the non-coding region of the gene would be of great interest as we attempt to learn the process of gene evolution.     

Conserved restriction sites within the ribosomal RNA genes of vertebrates

We have mapped the cleavage sites of four restriction enzymes which recognize six-base sequences within the nuclear ribosomal (rRNA) genes of twelve vertebrates, including several placental mammals (Homo sapiens, man; Bos taurus, cow; Equus caballus, horse; Sus scofra, pig; Ovis aries, sheep; Rattus rattus, rat), a marsupial (Didelphis marsupialis, opossum), a bird (Gallus domesticus, chicken), an amphibian (Xenopus laevis), a reptile (Alligator mississipiensis), a bony fish (Cynoscion nebulosus, sea trout), and a cartilagenous fish (Carcharhinus species, requiem shark). These animals represent a span of approx. 400 million years of evolutionary divergence. Our data identify restriction sites in the rRNA genes which are highly conserved among higher vertebrates and therefore are likely to be in functionally important regions. Additionally, the restriction enzyme sites identified will be useful in cloning and sequencing the rRNA genes in any vertebrate. Finally, the consistent size and conserved sequence homology suggests that these rRNA gene segments will be useful as internal controls in hybridization experiments involving other genomic regions in vertebrates.     

Sex chromosome homologies between human and fish revealed by the chromosome painting method

Human sex chromosome-specific probes were hybridized to metaphase spreads of three fish species, Monopterus albus Zuiew, Danio rerio and Mastacembelus aculeatus Basilewsky, to reveal evolutionary conservation of sex chromosomal segments between distantly related species of vertebrates. The human X chromosomal paint disclosed 4, 8, and 6 conserved syntenic segments in the karyotypes of the three fish species respectively, which were scattered in several pairs of homologous chromosomes. But no conserved segment was identified in our experiments when the human Y chromosomal probes were used. The evolution of the X chromosome of vertebrates is discussed.     

Presence of O6-methylguanine acceptor protein in the tissues of different classes of vertebrates and invertebrates

We have measured the ability of extracts of tissues from several species of mammals, birds, reptiles, amphibia and fish to demethylate adducts of O6-methylguanine in exogenous DNA by transfer of the methyl group to an acceptor protein. Our study also encompassed tissues from a smaller number of invertebrates, from arthropods, molluscs and annelids. The vertebrate tissues used were liver, brain, spleen and kidney. In the case of the invertebrates we sampled liver, neural tissue, gonads, digestive tract and hepatopancreas. There was no consistent change in the amount of acceptor activity per unit of protein or DNA going from cold-blooded to warm-blooded vertebrates. Liver invariably had the highest amount; this finding was not unexpected since metabolic processes in the liver are high, and good cellular protective mechanism important. Inter-class comparisons within the vertebrates are highly speculative, and hindered by the fact that there is little information on carcinogenesis in animals other than rodents and humans. O6-methylguanine acceptor activity was found in all the invertebrate tissues tested. The amounts were variable, 0.003-0.0051 fmol/micrograms cellular DNA, but the values fell within the range of those found in the tissues of vertebrates.     

EPR spin trapping of a radical intermediate in the urate oxidase reaction

Urate oxidase, or uricase (EC 1.7.3.3), is a peroxisomal enzyme that catalyses the oxidation of uric acid to allantoin. The chemical mechanism of the urate oxidase reaction has not been clearly established, but the involvement of radical intermediates was hypothesised. In this study EPR spectroscopy by spin trapping of radical intermediates has been used in order to demonstrate the eventual presence of radical transient urate species. The oxidation reaction of uric acid by several uricases (Porcine Liver, Bacillus Fastidiosus, Candida Utilitis) was performed in the presence of 5-diethoxyphosphoryl-5-methyl-pyrroline-N-oxide (DEPMPO) as spin trap. DEPMPO was added to reaction mixture and a radical adduct was observed in all cases. Therefore, for the first time, the presence of a radical intermediate in the uricase reaction was experimentally proved.     

EPR Spin Trapping of a Radical Intermediate in the Urate Oxidase Reaction

Urate oxidase, or uricase (EC 1.7.3.3), is a peroxisomal enzyme that catalyses the oxidation of uric acid to allantoin. The chemical mechanism of the urate oxidase reaction has not been clearly established, but the involvement of radical intermediates was hypothesised. In this study EPR spectroscopy by spin trapping of radical intermediates has been used in order to demonstrate the eventual presence of radical transient urate species. The oxidation reaction of uric acid by several uricases (Porcine Liver, Bacillus Fastidiosus, Candida Utilitis) was performed in the presence of 5‐diethoxyphosphoryl‐5‐methyl‐pyrroline‐N‐oxide (DEPMPO) as spin trap. DEPMPO was added to reaction mixture and a radical adduct was observed in all cases. Therefore, for the first time, the presence of a radical intermediate in the uricase reaction was experimentally proved.     

Sex Chromosomal Homologies between Human and Fish Karyotypes Revealed by Chromosome Painting

Human sex chromosome-specific probes were hybridized to metaphase spreads of three fish species, Monopterus albus Zuiew, Danio rerioandMastacembelus aculeatusBasilewsky, to reveal evolutionary conservation of sex chromosomal segments between distantly related species of vertebrates. The human X chromosomal paint disclosed 4, 8, and 6 conserved syntenic segments in the karyotypes of the three fish species respectively, which were scattered in several pairs of homologous chromosomes. But no conserved segment was identified in our experiments when the human Y chromosomal probes were used. The evolution of the X chromosome of vertebrates is discussed.