How nature tunes isoenzyme activity in the multifunctional catalytic globin dehaloperoxidase from Amphitrite ornata

The coelomic hemoglobin of Amphitrite ornata, termed dehaloperoxidase (DHP), is the first known multifunctional catalytic globin to possess biologically-relevant peroxidase and peroxygenase activities. Although the two isoenzymes of DHP, A and B, differ in sequence by only 5 amino acids out of 137 residues, DHP B consistently exhibits a greater activity than isoenzyme A. To delineate the contributions of each amino acid substitution to the activity of either isoenzyme, the substitutions of the five amino acids were systematically investigated, individually and in combination, using 22 mutants. Biochemical assays and mechanistic studies demonstrated that the mutants that only contained the I9L substitution showed increased i) k_cat values (peroxidase activity), ii) 5-Br-indole conversion and binding affinity (peroxygenase activity), and iii) rate of Compound ES formation (enzyme activation). Whereas the X-ray structures of the oxyferrous forms of DHP B (L9I) (1.96 Å), DHP A (I9L) (1.20 Å), and WT DHP B (1.81 Å) showed no significant differences, UV–visible spectroscopy (A_Soret/A₃₈₀ ratio) revealed that the I9L substitution increased the 5-coordinate high-spin heme population characterized by the “open” conformation (i.e., distal histidine swung out of the pocket), which likely favors substrate binding. The positioning of the distal histidine closer to the heme cofactor in the solution state also appears to facilitate activation of DHP via the Compound ES intermediate. Taken together, the studies undertaken here shed light on the structure-function relationship in dehaloperoxidase, but also help to establish the foundation for understanding how enzymatic activity can be tuned in isoenzymes of a multifunctional catalytic globin.     

Degradation of sulfide by dehaloperoxidase-hemoglobin from <Emphasis Type="Italic">Amphitrite ornata</Emphasis>

Dehaloperoxidase-hemoglobin (DHP) is a unique multifunctional enzyme with a globin fold. The enzyme serves as the respiratory hemoglobin for the marine worm Amphitrite ornata and has been shown to catalyze the conversion of highly toxic trihalophenols to dihaloquinones as a detoxification function for the organism. Given the simplicity of the structure of A. ornata, it is entirely possible that DHP may play an even more general role in detoxification of the organism from sulfide commonly found in the coastal estuaries where A. ornata thrives. Comparison of DHP with other sulfide-binding hemoglobins shows that DHP possesses several distal cavity structural properties, such as an aromatic cage and a hydrogen-bond-donor amino acid (His55), that facilitate sulfide binding. Furthermore, a complete reduction of the ferric heme occurs after sulfide exposure under aerobic or anaerobic conditions to yield either the oxy or the deoxy ferrous states of DHP, respectively. Oxidation of sulfide by the heme leads to sulfur products that are less toxic to A. ornata. This proposed new function for DHP relies on the highly flexible distal His55 for deprotonation of the bound hydrogen sulfide, similar to H₂O₂ activation of the peroxidase function, and provides further support for the importance of the flexibility of the distal His55 in this novel globin.     

Oxidative dechlorination of halogenated phenols catalyzed by two distinct enzymes: Horseradish peroxidase and dehaloperoxidase

The mechanism of the dehalogenation step catalyzed by dehaloperoxidase (DHP) from Amphitrite ornata, an unusual heme-containing protein with a globin fold and peroxidase activity, has remarkable similarity with that of the classical heme peroxidase, horseradish peroxidase (HRP). Based on quantum mechanical/molecular mechanical (QM/MM) modeling and experimentally determined chlorine kinetic isotope effects, we have concluded that two sequential one electron oxidations of the halogenated phenol substrate leads to a cationic intermediate that strongly resembles a Meisenheimer intermediate – a commonly formed reactive complex during nucleophilic aromatic substitution reactions especially in the case of arenes carrying electron withdrawing groups.     

Characterization of Two Alcohol Dehydrogenase (Adh) Loci from the Olive Fruit Fly, Bactrocera (Dacus) oleae and Implications for Adh Duplication in Dipteran Insects

We report the cloning and structural characterization of two Adh loci of the olive fruit fly, Bactrocera oleae. Each of the two genes, named Adh1 and Adh2, consists of three exons and two introns for a total length of 1981 and 988 nucleotides, respectively. Their deduced amino acid sequences of 257 and 258 residues exhibit a 77% identity and display the characteristics of the insect ADH enzymes, which belong to the short-chain dehydrogenases/reductases family. The Adh genes of B. oleae are compared to the two genes of the Mediterranean fly, Ceratitis capitata, the only other species of the Tephritidae family in which the Adh genes have been studied. On the basis of amino acid divergence the four genes form two clusters each containing one gene from each species, as expected if there was one duplication event before speciation. On the basis of nucleotide sequence the four sequences form two clusters each containing the two sequences from the same species, as expected if there was a separate duplication event in each species. To help decide between the two alternatives, we compared at both the amino acid and DNA level the Adh genes of five Drosophila species that are known to carry two such genes and observed that, with only one exception at the amino acid level, conspecific loci cluster together. We conclude that the information we have at present does not allow a firm choice between the hypothesis of a single duplication event that occurred before the split of Bactrocera and Ceratitis from their common ancestor and the hypothesis of two independent duplication events, one in each of the two genera. Received: 30 May 2000 / Accepted: 17 August 2000     

The crystal structure and amino acid sequence of dehaloperoxidase from Amphitrite ornata indicate common ancestry with globins

The full-length, protein coding sequence for dehaloperoxidase was obtained using a reverse genetic approach and a cDNA library from marine worm Amphitrite ornata. The crystal structure of the dehaloperoxidase (DHP) was determined by the multiple isomorphous replacement method and was refined at 1.8-A resolution. The enzyme fold is that of the globin family and, together with the amino acid sequence information, indicates that the enzyme evolved from an ancient oxygen carrier. The peroxidase activity of DHP arose mainly through changes in the positions of the proximal and distal histidines relative to those seen in globins. The structure of a complex of DHP with 4-iodophenol is also reported, and it shows that in contrast to larger heme peroxidases DHP binds organic substrates in the distal cavity. The binding is facilitated by the histidine swinging in and out of the cavity. The modeled position of the oxygen atom bound to the heme suggests that the enzymatic reaction proceeds via direct attack of the oxygen atom on the carbon atom bound to the halogen atom.     

Globin gene family evolution and functional diversification in annelids

Globins are the most common type of oxygen-binding protein in annelids. In this paper, we show that circulating intracellular globin (Alvinella pompejana and Glycera dibranchiata), noncirculating intracellular globin (Arenicola marina myoglobin) and extracellular globin from various annelids share a similar gene structure, with two conserved introns at canonical positions B12.2 and G7.0. Despite sequence divergence between intracellular and extracellular globins, these data strongly suggest that these three globin types are derived from a common ancestral globin-like gene and evolved by duplication events leading to diversification of globin types and derived functions. A phylogenetic analysis shows a distinct evolutionary history of annelid extracellular hemoglobins with respect to intracellular annelid hemoglobins and mollusc and arthropod extracellular hemoglobins. In addition, dehaloperoxidase (DHP) from the annelid, Amphitrite ornata, surprisingly exhibits close phylogenetic relationships to some annelid intracellular globins. We have characterized the gene structure of A. ornata DHP to confirm assumptions about its homology with globins. It appears that it has the same intron position as in globin genes, suggesting a common ancestry with globins. In A. ornata, DHP may be a derived globin with an unusual enzymatic function.     

Ribosomal Protein S1(RpsA) of Buchnera aphidicola,the Endosymbiont of Aphids: Characterization of the Gene and Detection of the Product

Buchnera aphidicola is a prokaryotic endosymbiont found in specialized cells of the aphid Schizaphis graminum. Many of the previously cloned B. aphidicola genes are preceded by a poor ribosome-binding site. Ribosomal protein S1 (RpsA) allows the translation of messenger RNAs that lack or have a poor ribosome binding site. We have cloned and sequenced a 4.5-kilobase (kb) B. aphidicola DNA fragment containing four open reading frames corresponding to aroA–rpsA–himD–tpiA. The deduced amino acid sequence of B. aphidicola RpsA was 75% identical to that of the Escherichia coli protein. The major difference was in the number of basic amino acids, which were present in higher numbers in B. aphidicola RpsA. Antiserum to E. coli RpsA was prepared and used to detect B. aphidicola RpsA in cell-free extracts of aphids. During the first 12 days of aphid growth there is a slight decrease in the amount of RpsA per unit of aphid weight. The three additional genes found on the 4.5-kb DNA fragment encoded for proteins involved in aromatic amino acid biosynthesis (aroA), DNA bending (himD), and carbohydrate metabolism (tpiA). The presence of these genes in B. aphidicola is additional evidence of its similarity to free-living bacteria.     

Four genes in two diverged subfamilies encode the ribulose-1,5-bisphosphate carboxylase small subunit polypeptides of Arabidopsis thaliana

The multigene family encoding the small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase/oxygenase in the crucifer Arabidopsis thaliana has been isolated and the organization and structure of the individual members determined. The family consists of four genes which have been divided into two subfamilies on the basis of linkage and DNA and amino acid sequence similarities. Three of the genes, designated ats1B, ats2B, and ats3B, reside in tandem on an 8 kb stretch of the chromosome. These genes share greater than 95% similarity in DNA sequence and encode polypeptides identical in length and 96.7% similar in amino acid sequence. The fourth gene, ats1A, is at least 10 kb removed from, or completely unlinked to the B subfamily. The B subfamily genes are more similar to each other than to ats1A in nucleotide and amino acid sequence. All four genes are interupted by two introns whose placement within the coding region of the genes is conserved. The introns of the B subfamily genes are similar in length and nucleotide sequence, but show no similarity to the introns of ats1A. Comparison of the DNA sequences within the immediate 5 and 3 flanking sequences among the genes revealed only limited regions of homology. S1 analysis shows that all four genes are expressed.     

The identification, cloning and mutagenesis of a genetic locus required for lipopolysaccharide biosynthesis in Bordetella pertussis

Bordetella pertussis lipopolysaccharide (LPS) is biologically active, being both toxic and immunogenic. Using transposon mutagenesis we have identified a genetic locus required for the biosynthesis of LPS in B. pertussis, which has been cloned and sequenced. We have also identified equivalent loci in Bordetella bronchiseptica and Bordetella parapertussis and cloned part of it from B. parapertussis. The amino acid sequences derived from most of the genes present in the sequenced B. pertussis locus are similar to proteins required for the biosynthesis of LPS and other complex polysaccharides from a variety of bacteria. The genes are in a unique arrangement in the locus. Several of the genes identified are similar to genes previously shown to play specific roles in LPS O-antigen biosynthesis. In particular, the amino acid sequence derived from one of the genes is similar to the enzyme encoded by rfbP from Salmonella enterica, which catalyses the transfer of galactose to the undecaprenol phosphate antigen carrier lipid as the first step in building oligosaccharide O-antigen units, which are subsequently assembled to form polymerized O-antigen structures. Defined mutation of this gene in the B. pertussis chromosome results in the inability to express band A LPS, possibly suggesting that the trisaccharide comprising band A is a single O-antigen-like structure and that B. pertussis LPS is similar to semi-rough LPS seen in some mutants of enteric bacteria.     

Cloning of Complementary and Genomic DNAs Encoding Echotoxins,Proteinaceous Toxins from the Salivary Gland of Marine Gastropod <Emphasis Type="Italic">Monoplex echo</Emphasis>

Echotoxins are hemolytic and lethal proteinaceous toxins of about 25 kDa that are contained in the salivary gland of marine gastropod Monoplex echo. In this study, complementary DNAs (cDNAs) encoding four echotoxins (2, A, B1 and B2) were isolated from the cDNA library constructed from the M. echo salivary gland and completely sequenced. Although the amino acid sequence identity between the four echotoxins and actinoporins (20 kDa hemolysins from sea anemones) was very low (12–16%), some amino acid residues important for the biological activity of actinoporins were well conserved in the echotoxins. In the case of echotoxin 2, the genomic DNA corresponding to the coding region was amplified. Sequencing data revealed that the echotoxin 2 gene is devoid of introns within the coding region as reported for the actinoporin genes. These results suggest that echotoxins have evolved from actinoporins or both toxins have evolved from the same ancestor.     

Molecular cloning,characterization and expression analysis of two catalase isozyme genes in barley

Clones representing two distinct barley catalase genes, Cat1 and Cat2, were found in a cDNA library prepared from seedling polysomal mRNA. Both clones were sequenced, and their deduced amino acid sequences were found to have high homology with maize and rice catalase genes. Cat1 had a 91% deduced amino acid sequence identity to CAT-1 of maize and 92% to CAT B of rice. Cat2 had 72 and 79% amino acid sequence identities to maize CAT-2 and-3 and 89% to CAT A of rice. Barley, maize or rice isozymes could be divided into two distinct groups by amino acid homologies, with one group homologous to the mitochondria-associated CAT-3 of maize and the other homologous to the maize peroxisomal/glyoxysomal CAT-1. Both barley CATs contained possible peroxisomal targeting signals, but neither had favorable mitochondrial targeting sequences. Cat1 mRNA occurred in whole endosperms (aleurones plus starchy endosperm), in isolated aleurones and in developing seeds, but Cat2 mRNA was virtually absent. Both mRNAs displayed different developmental expression patterns in scutella of germinating seeds. Cat2 mRNA predominated in etiolated seedling shoots and leaf blades. Barley genomic DNA contained two genes for Cat1 and one gene for Cat2. The Cat2 gene was mapped to the long arm of chromosome 4, 2.9 cM in telomeric orientation from the mlo locus conferring resistance to the powdery mildew fungus (Erysiphe graminis f.sp. hordei).     

Tyrosyl Radicals in Dehaloperoxidase: HOW NATURE DEALS WITH EVOLVING AN OXYGEN-BINDING GLOBIN TO A BIOLOGICALLY RELEVANT PEROXIDASE*

Dehaloperoxidase (DHP) from Amphitrite ornata, having been shown to catalyze the hydrogen peroxide-dependent oxidation of trihalophenols to dihaloquinones, is the first oxygen binding globin that possesses a biologically relevant peroxidase activity. The catalytically competent species in DHP appears to be Compound ES, a reactive intermediate that contains both a ferryl heme and a tyrosyl radical. By simulating the EPR spectra of DHP activated by H₂O₂, Thompson et al. (Thompson, M. K., Franzen, S., Ghiladi, R. A., Reeder, B. J., and Svistunenko, D. A. (2010) J. Am. Chem. Soc. 132, 17501–17510) proposed that two different radicals, depending on the pH, are formed, one located on either Tyr-34 or Tyr-28 and the other on Tyr-38. To provide additional support for these simulation-based assignments and to deduce the role(s) that tyrosyl radicals play in DHP, stopped-flow UV-visible and rapid-freeze-quench EPR spectroscopic methods were employed to study radical formation in DHP when three tyrosine residues, Tyr-28, Tyr-34, and Tyr-38, were replaced either individually or in combination with phenylalanines. The results indicate that radicals form on all three tyrosines in DHP. Evidence for the formation of DHP Compound I in several tyrosine mutants was obtained. Variants that formed Compound I showed an increase in the catalytic rate for substrate oxidation but also an increase in heme bleaching, suggesting that the tyrosines are necessary for protecting the enzyme from oxidizing itself. This protective role of tyrosines is likely an evolutionary adaptation allowing DHP to avoid self-inflicted damage in the oxidative environment.     

Phylogeny,genetic diversity and phylogeography of the genus Codoma (Teleostei,Cyprinidae)

This study represents a thorough analysis of Codoma, a monotypic genus endemic to north‐western Mexico. A previous morphological analysis of the species concluded that there exists several morphological groups in Codoma ornata, suggesting diversity in Codoma could be underestimated. No studies have examined the genetic diversity in Codoma ornata to test this hypothesis and identify independent lineages. We present a phylogeographic analysis using one mitochondrial and two nuclear genes, and specimens from across nine major drainages in both the Chihuahuan Desert and the Sierra Madre Occidental of western Mexico. All genes and analyses recovered populations of Codoma in a well‐supported clade and sister to Tampichthys, and this clade sister to Cyprinella. Analyses of both mitochondrial and nuclear genes indicated Codoma is not monotypic and recover more diversity in the genus than currently recognized. The four (mitochondrial) and five (nuclear) genetically distinct lineages are consistent with those groups outlined in the prior morphological study of the genus. Composition and distribution of these major lineages is also consistent with prior biogeographic hypothesis for other fishes in the region, supporting an ancestral Rio Grande system extending south towards central Mexico. Fragmentation of this paleosystem was followed by allopatric speciation in the Chihuahuan Desert. These results suggest a scenario of long‐term isolation in four major regions (upper Conchos, lower Conchos, Nazas, upper Mezquital). Resolution of the diversity and biogeography of these lineages has many implications for various biological disciplines, especially for evolutionary and conservation studies.     

Identification and Cloning of Peptide Synthetase Genes of Thermostable Bacilli Using the Polymerase Chain Reaction

Fourteen thermophilic and thermostable strains of the genus Bacillus were studied. Total DNA was isolated from these strains and used as a template to identify and clone peptide synthetase genes by means of polymerase chain reaction. Amplified DNA fragments were cloned into a phasmid vector, and nucleotide sequences of cloned fragments were determined. Stringent thermophilic strains were shown to lack genetic systems, which are responsible for the synthesis of secondary metabolites and homologous to the known peptide synthetase genes. On the contrary, thermostable strains had peptide synthetases and produced antimicrobial secondary metabolites. Analysis of nucleotide sequences and deduced amino acid sequences of cloned PCR fragments from B. licheniformis strains VK2, VK21, and VK2101 showed that they are absolutely identical. The cloned DNA fragment was found to be a portion of the open reading frame, which we termed ORF1. Data from analysis of a partial nucleotide sequence of the peptide synthetase gene of strain VK21 indicated that a 9.5-kb region of chromosomal DNA contains sequences of two genes homologous to the B. subtilis peptide synthetase genesdhbB and dhbF. Strains VK2, VK21, and VK2101 were shown to synthesize siderophores. A method for screening bacteria with peptide synthetase genes has been developed.     

Phylogenetic analysis of archaeal PCNA homologues

Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domain Eucarya. Eukaryotes and euryarchaeotes, which belong to one subdomain of Archaea, possess a single PCNA homologue, whereas two distinct PCNA homologues have been identified from Sulfolobus solfataricus, which belongs to the other archaeal subdomain, Crenarchaeota. We have cloned and sequenced two genes of PCNA homologues from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis. These genes, referred to as the Soh PCNA A gene and the Soh PCNA B gene, were found to encode 245 amino acids (aa) (27 kDa) and 248 aa (27 kDa), respectively. In deduced amino acid sequences of both PCNA homologues, the motif L/I-A-P-K/R, implicated in binding of PCNA with replication factor C (RFC), was identified. Phylogenetic analysis of all available archaeal PCNA homologues suggests that crenarchaeal homologues are divided into two groups. Group A consists of Soh PCNA A, one of the S. solfataricus PCNA homologues, and one of the Aeropyrum pernix PCNA homologues. The other crenarchaeal homologues form group B. Crenarchaeal PCNA homologues constitute a monophyletic subfamily. These results suggest that the evolution of crenarchaeal PCNA homologues has been characterized by one or two gene duplication events, which are assumed to have occurred after the split of the crenarchaeal and euryarchaeal lineages. Received: July 10, 2000 / Accepted: September 26, 2000     

The myrosinase (thioglucoside glucohydrolase) gene family in Brassicaceae

The glucosinolate hydrolyzing enzymes myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) are encoded by a multigene family consisting of two subgroups. The first two nuclear genes representing each of these two subgroups of the new gene family, Myr1.Bn1 and Myr2.Bn1, from Brassica napus have been cloned and sequenced. Based on conserved regions in cDNA of three species, PCR (polymerase chain reaction) primers were made, and used to amplify and characterize the structure of the myrosinase genes in seven species of Brassiceae. Southern hybridization analysis of PCR products and genomic DNA indicates that myrosinase is encoded by at least 14 genes in B. napus, with similar numbers in the other species of Brassicaceae investigated. The Myr1 gene cloned from B. napus has a 19 amino acid signal peptide and consists of 11 exons of sizes ranging from 54 to 256 bp and 10 introns of sizes from 75 to 229 bp. The Myr2 gene has a 20 amino acid signal peptide and consists of 12 exons ranging in size from 35 to 262 bp and 11 introns of sizes from 81 to 131 bp. The exons from the two genes have 83% homology at the amino acid level. The intron-exon splice sites are of GT..AG consensus type. The signal peptides and presence of sites for N-linked glycosylation, suggest transport and glycosylation through the ER-Golgi complex. The differences between the two genes are discussed on the basis of their predicted expression at different developmental stages in the plant. Both genes show homology to a conserved motif representing the glycosyl hydrolase family of enzymes.     

Purification of chloroplast elongation factor Tu and cDNA analysis in tobacco: the existence of two chloroplast elongation factor Tu species

We have purified a chloroplast elongation factor Tu (EF-Tu) from tobacco (Nicotiana tabacum) and determined its N-terminal amino acid sequence. Two distinct cDNAs encoding EF-Tu were isolated from a leaf cDNA library of N. sylvestris (the female progenitor of N. tabacum) using an oligonucleotide probe based on the EF-Tu protein sequence. The cDNA sequence and genomic Southern analyses revealed that tobacco chloroplast EF-Tu is encoded by two distinct genes in the nuclear genome of N. sylvestris. We designated the corresponding gene products EF-Tu A and B. The mature polypeptides of EF-Tu A and B are 408 amino acids long and share 95.3% amino acid identity. They show 75–78% amino acid identity with cyanobacterial and chloroplast-encoded EF-Tu species.     

DNA sequence analysis of the recA genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r

Summary The complete nucleotide sequences of therecA genes fromEscherichia coli B/r,Shigella flexneri, Erwinia carotovora andProteus vulgaris were determined. The DNA sequence of the coding region of theE. coli B/r gene contained a single nucleotide change compared with theE. coli K12 gene sequence whereas theS. flexneri gene differed at 7 residues. In both cases, the predicted proteins were identical in primary structure to theE. coli K12 RecA protein. The DNA sequences of the recA genes fromE. carotovora andP. vulgaris were 80% and 74% homologous, respectively, to theE. coli K12 gene. The predicted amino acid sequences of theE. carotovora andP. vulgaris RecA proteins were 91% and 85% identical respectively, to that ofE. coli K12. The RecA proteins from bothP. vulgaris andE. carotovora diverged significantly in sequence in the last 50 residues whereas they showed striking conservation throughout the first 300 amino acids which include an ATP-binding region and a subunit interaction domain. A putative LexA repressor binding site was localized upstream of each of the heterologous genes.     

Identification of mucAB-like homologs on two IncT plasmids, R394 and Rts-1

Recent phylogenetic analysis of the superfamily of lesion-replicating DNA polymerases suggest that they can be broadly divided into four sub-groups comprised of UmuC-like, DinB-like, Rev1-like and Rad30-like proteins. The UmuC-like sub-family is best characterized at the genetic level and sequence analysis of eleven umu orthologs, residing on bacterial chromosomes or on self-transmissible R-plasmids allows further subdivision into five sub-groups (UmuDC, MucAB, ImpAB, RumAB and RulAB) based on amino acid sequence conservation. Some of these orthologs are apparently inactive in situ, but may promote increased mutagenesis and survival when subcloned and expressed from high-copy number plasmids. We were, therefore, interested in devising an assay that would identify umuC-like genes in situ in the absence of a functional assay. To this end, degenerate primers directed towards conserved amino acid regions within the UmuC-like sub-family of DNA polymerases were designed and used to identify mucAB-like operons on the IncT plasmids, R394 and Rts-1.Interestingly, DNA sequence analysis of an 7 kb region of R394 identified two LexA-regulated genes immediately downstream of mucAB_(R394) that are similar to the chromosomally-encoded Escherichia coli tus gene and the IncI plasmid-encoded impC gene, respectively. Analysis of the R394 and Rts-1 mucB genes revealed that both contain insertions which result in the expression of a truncated inactive MucB protein. While R394 was unable to restore mutagenesis functions to a ΔumuDC E. coli strain, Rts-1 surprisingly promoted significant levels of MMS-induced SOS mutagenesis, raising the possibility that Rts-1 encodes another, yet unidentified, umu-like homolog.