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Archives of Microbiology (v.185, #1)
Beta-propeller phytases in the aquatic environment by Chiwai Cheng; Boon L. Lim (pp. 1-13).
Phytate, which is one of the dominant organic phosphorus compounds in nature, is very stable in soils. Although a substantial amount of phytate is carried from terrestrial to aquatic systems, it is a minor component of organic phosphorus in coastal sediments. The ephemeral nature of phytate implies the rapid hydrolysis of phytate under aquatic conditions. Among the four classes of known phytases that have been identified in terrestrial organisms, only β-propeller phytase-like sequences have been identified in the aquatic environment. A novel β-propeller phytase gene (phyS), cloned from Shewanella oneidensis MR-1, was found to encode a protein with two beta-propeller phytase domains. The characterization of recombinant full-length PhyS and its domains demonstrated that Domain II was the catalytic domain responsible for phytate hydrolysis. The full-length PhyS displayed a Km of 83 μM with a kcat of 175.9 min−1 and the Domain II displayed a Km of 474 μM with a kcat of 10.6 min−1. These results confirm that the phyS gene encodes a functional β-propeller phytase, which is expressed in S. oneidensis under phosphorus deficienct condition. The presence of multiple sequences with a high similarity to phyS in aquatic environmental samples and the widespread occurrence of the Shewanella species in nature suggest that the β-propeller phytase family is the major class of phytases in the aquatic environment, and that it may play an important role in the recycling of phosphorus.
Keywords: Aquatic; Beta-propeller; Shewanella oneidensis ; Phosphorus; Phytase; Phytate
Beta-propeller phytases in the aquatic environment by Chiwai Cheng; Boon L. Lim (pp. 1-13).
Phytate, which is one of the dominant organic phosphorus compounds in nature, is very stable in soils. Although a substantial amount of phytate is carried from terrestrial to aquatic systems, it is a minor component of organic phosphorus in coastal sediments. The ephemeral nature of phytate implies the rapid hydrolysis of phytate under aquatic conditions. Among the four classes of known phytases that have been identified in terrestrial organisms, only β-propeller phytase-like sequences have been identified in the aquatic environment. A novel β-propeller phytase gene (phyS), cloned from Shewanella oneidensis MR-1, was found to encode a protein with two beta-propeller phytase domains. The characterization of recombinant full-length PhyS and its domains demonstrated that Domain II was the catalytic domain responsible for phytate hydrolysis. The full-length PhyS displayed a Km of 83 μM with a kcat of 175.9 min−1 and the Domain II displayed a Km of 474 μM with a kcat of 10.6 min−1. These results confirm that the phyS gene encodes a functional β-propeller phytase, which is expressed in S. oneidensis under phosphorus deficienct condition. The presence of multiple sequences with a high similarity to phyS in aquatic environmental samples and the widespread occurrence of the Shewanella species in nature suggest that the β-propeller phytase family is the major class of phytases in the aquatic environment, and that it may play an important role in the recycling of phosphorus.
Keywords: Aquatic; Beta-propeller; Shewanella oneidensis ; Phosphorus; Phytase; Phytate
Betaine aldehyde dehydrogenase from Pseudomonas aeruginosa: cloning, over-expression in Escherichia coli, and regulation by choline and salt by Roberto Velasco-García; Miguel Angel Villalobos; Miguel A. Ramírez-Romero; Carlos Mújica-Jiménez; Gabriel Iturriaga; Rosario A. Muñoz-Clares (pp. 14-22).
In the human pathogen Pseudomonas aeruginosa, betaine aldehyde dehydrogenase (BADH) may play a dual role assimilating carbon and nitrogen from choline or choline precursors—abundant at infection sites—and producing glycine betaine, which protects the bacteria against the high-osmolarity stress prevalent in the infected tissues. We cloned the P. aeruginosa BADH gene and expressed the BADH protein in Escherichia coli. The recombinant protein appears identical to its native counterpart, as judged by Western blot, N-terminal amino acid sequence, tryptophan-fluorescence emission spectra, circular-dichroism spectroscopy, size-exclusion chromatography, and kinetic properties. Computational analysis indicated that the promoter sequence of the putative operon that includes the BADH gene has a consensus-binding site for the choline-sensing transcription repressor BetI, and putative boxes for ArcA and Lrp transcription factors but no known elements of response to osmotic stress. This is consistent with the strong induction of BADH expression by choline and with the lack of effect of NaCl. As there were significant amounts of BADH protein and activity in P. aeruginosa cells grown on glucose plus choline, as well as the BADH activity exhibiting tolerance to salt, it is likely that glycine betaine is synthesized in vivo and could play an important osmoprotectant role under conditions of infection.
Keywords: Pseudomonas aeruginosa ; Betaine aldehyde dehydrogenase; Bet system; Computational promoter analysis; Choline induction; Salt inhibition
Betaine aldehyde dehydrogenase from Pseudomonas aeruginosa: cloning, over-expression in Escherichia coli, and regulation by choline and salt by Roberto Velasco-García; Miguel Angel Villalobos; Miguel A. Ramírez-Romero; Carlos Mújica-Jiménez; Gabriel Iturriaga; Rosario A. Muñoz-Clares (pp. 14-22).
In the human pathogen Pseudomonas aeruginosa, betaine aldehyde dehydrogenase (BADH) may play a dual role assimilating carbon and nitrogen from choline or choline precursors—abundant at infection sites—and producing glycine betaine, which protects the bacteria against the high-osmolarity stress prevalent in the infected tissues. We cloned the P. aeruginosa BADH gene and expressed the BADH protein in Escherichia coli. The recombinant protein appears identical to its native counterpart, as judged by Western blot, N-terminal amino acid sequence, tryptophan-fluorescence emission spectra, circular-dichroism spectroscopy, size-exclusion chromatography, and kinetic properties. Computational analysis indicated that the promoter sequence of the putative operon that includes the BADH gene has a consensus-binding site for the choline-sensing transcription repressor BetI, and putative boxes for ArcA and Lrp transcription factors but no known elements of response to osmotic stress. This is consistent with the strong induction of BADH expression by choline and with the lack of effect of NaCl. As there were significant amounts of BADH protein and activity in P. aeruginosa cells grown on glucose plus choline, as well as the BADH activity exhibiting tolerance to salt, it is likely that glycine betaine is synthesized in vivo and could play an important osmoprotectant role under conditions of infection.
Keywords: Pseudomonas aeruginosa ; Betaine aldehyde dehydrogenase; Bet system; Computational promoter analysis; Choline induction; Salt inhibition
The DD-carboxypeptidase activity encoded by pbp4B is not essential for the cell growth of Escherichia coli by Daniel Vega; Juan A. Ayala (pp. 23-27).
The gene (pbp4B) encoding a putative DD-carboxypeptidase has been deleted in Escherichia coli and it is shown to be not essential for cell division. Disruption of the gene in a genetic background where all putative activities of DD-carboxypeptidases and/or DD-endopeptidases had been eliminated indicates that these activities are not required for cell growth in enterobacteria. The penicillin-binding capacity and a low DD-carboxypeptidase activity of PBP4B are demonstrated.
Keywords: Penicillin-binding proteins; DD-carboxypeptidase; Cell division; Escherichia coli
The DD-carboxypeptidase activity encoded by pbp4B is not essential for the cell growth of Escherichia coli by Daniel Vega; Juan A. Ayala (pp. 23-27).
The gene (pbp4B) encoding a putative DD-carboxypeptidase has been deleted in Escherichia coli and it is shown to be not essential for cell division. Disruption of the gene in a genetic background where all putative activities of DD-carboxypeptidases and/or DD-endopeptidases had been eliminated indicates that these activities are not required for cell growth in enterobacteria. The penicillin-binding capacity and a low DD-carboxypeptidase activity of PBP4B are demonstrated.
Keywords: Penicillin-binding proteins; DD-carboxypeptidase; Cell division; Escherichia coli
Bacterial type III polyketide synthases: phylogenetic analysis and potential for the production of novel secondary metabolites by heterologous expression in pseudomonads by Frank Gross; Nora Luniak; Olena Perlova; Nikolaos Gaitatzis; Holger Jenke-Kodama; Klaus Gerth; Daniela Gottschalk; Elke Dittmann; Rolf Müller (pp. 28-38).
Type III polyketide synthases (PKS) were regarded as typical for plant secondary metabolism before they were found in microorganisms recently. Due to microbial genome sequencing efforts, more and more type III PKS are found, most of which of unknown function. In this manuscript, we report a comprehensive analysis of the phylogeny of bacterial type III PKS and report the expression of a type III PKS from the myxobacterium Sorangium cellulosum in pseudomonads. There is no precedent of a secondary metabolite that might be biosynthetically correlated to a type III PKS from any myxobacterium. Additionally, an inactivation mutant of the S. cellulosum gene shows no physiological difference compared to the wild-type strain which is why these type III PKS are assumed to be “silent” under the laboratory conditions administered. One type III PKS (SoceCHS1) was expressed in different Pseudomonas sp. after the heterologous expression in Escherichia coli failed. Cultures of recombinant Pseudomonas sp. harbouring SoceCHS1 turned red upon incubation and the diffusible pigment formed was identified as 2,5,7-trihydroxy-1,4-naphthoquinone, the autooxidation product of 1,3,6,8-tetrahydroxynaphthalene. The successful heterologous production of a secondary metabolite using a gene not expressed under administered laboratory conditions provides evidence for the usefulness of our approach to activate such secondary metabolite genes for the production of novel metabolites.
Keywords: Myxobacteria; Type III PKS; Silent gene; Pseudomonads; Flaviolin; Heterologous Expression; Genome sequencing; Phylogeny
Bacterial type III polyketide synthases: phylogenetic analysis and potential for the production of novel secondary metabolites by heterologous expression in pseudomonads by Frank Gross; Nora Luniak; Olena Perlova; Nikolaos Gaitatzis; Holger Jenke-Kodama; Klaus Gerth; Daniela Gottschalk; Elke Dittmann; Rolf Müller (pp. 28-38).
Type III polyketide synthases (PKS) were regarded as typical for plant secondary metabolism before they were found in microorganisms recently. Due to microbial genome sequencing efforts, more and more type III PKS are found, most of which of unknown function. In this manuscript, we report a comprehensive analysis of the phylogeny of bacterial type III PKS and report the expression of a type III PKS from the myxobacterium Sorangium cellulosum in pseudomonads. There is no precedent of a secondary metabolite that might be biosynthetically correlated to a type III PKS from any myxobacterium. Additionally, an inactivation mutant of the S. cellulosum gene shows no physiological difference compared to the wild-type strain which is why these type III PKS are assumed to be “silent” under the laboratory conditions administered. One type III PKS (SoceCHS1) was expressed in different Pseudomonas sp. after the heterologous expression in Escherichia coli failed. Cultures of recombinant Pseudomonas sp. harbouring SoceCHS1 turned red upon incubation and the diffusible pigment formed was identified as 2,5,7-trihydroxy-1,4-naphthoquinone, the autooxidation product of 1,3,6,8-tetrahydroxynaphthalene. The successful heterologous production of a secondary metabolite using a gene not expressed under administered laboratory conditions provides evidence for the usefulness of our approach to activate such secondary metabolite genes for the production of novel metabolites.
Keywords: Myxobacteria; Type III PKS; Silent gene; Pseudomonads; Flaviolin; Heterologous Expression; Genome sequencing; Phylogeny
Physiological role of d-amino acid-N-acetyltransferase of Saccharomyces cerevisiae: detoxification of d-amino acids by Geok-Yong Yow; Takuma Uo; Tohru Yoshimura; Nobuyoshi Esaki (pp. 39-46).
Saccharomyces cerevisiae is sensitive to d-amino acids: those corresponding to almost all proteinous l-amino acids inhibit the growth of yeast even at low concentrations (e.g. 0.1 mM). We have determined that d-amino acid-N-acetyltransferase (DNT) of the yeast is involved in the detoxification of d-amino acids on the basis of the following findings. When the DNT gene was disrupted, the resulting mutant was far less tolerant to d-amino acids than the wild type. However, when the gene was overexpressed with a vector plasmid p426Gal1 in the wild type or the mutant S. cerevisiae as a host, the recombinant yeast, which was found to show more than 100 times higher DNT activity than the wild type, was much more tolerant to d-amino acids than the wild type. We further confirmed that, upon cultivation with d-phenylalanine, N-acetyl-d-phenylalanine was accumulated in the culture but not in the wild type and hpa3Δ cells overproducing DNT cells. Thus, d-amino acids are toxic to S. cerevisiae but are detoxified with DNT by N-acetylation preceding removal from yeast cells.
Keywords: Saccharomyces cerevisiae ; d-amino acids; Toxicity; d-amino acid-N-acetyltransferase
Physiological role of d-amino acid-N-acetyltransferase of Saccharomyces cerevisiae: detoxification of d-amino acids by Geok-Yong Yow; Takuma Uo; Tohru Yoshimura; Nobuyoshi Esaki (pp. 39-46).
Saccharomyces cerevisiae is sensitive to d-amino acids: those corresponding to almost all proteinous l-amino acids inhibit the growth of yeast even at low concentrations (e.g. 0.1 mM). We have determined that d-amino acid-N-acetyltransferase (DNT) of the yeast is involved in the detoxification of d-amino acids on the basis of the following findings. When the DNT gene was disrupted, the resulting mutant was far less tolerant to d-amino acids than the wild type. However, when the gene was overexpressed with a vector plasmid p426Gal1 in the wild type or the mutant S. cerevisiae as a host, the recombinant yeast, which was found to show more than 100 times higher DNT activity than the wild type, was much more tolerant to d-amino acids than the wild type. We further confirmed that, upon cultivation with d-phenylalanine, N-acetyl-d-phenylalanine was accumulated in the culture but not in the wild type and hpa3Δ cells overproducing DNT cells. Thus, d-amino acids are toxic to S. cerevisiae but are detoxified with DNT by N-acetylation preceding removal from yeast cells.
Keywords: Saccharomyces cerevisiae ; d-amino acids; Toxicity; d-amino acid-N-acetyltransferase
Utilization of homoserine lactone as a sole source of carbon and energy by soil Arthrobacter and Burkholderia species by Wan-Wan Yang; Jong-In Han; Jared Renton Leadbetter (pp. 47-54).
Homoserine lactone (HSL) is a ubiquitous product of metabolism. It is generated by all known biota during the editing of certain mischarged aminoacyl-tRNA reactions, and is also released as a product of quorum signal degradation by bacterial species expressing acyl-HSL acylases. Little is known about its environmental fate over long or short periods of time. The mammalian enzyme paraoxonase, which has no known homologs in bacteria, has been reported to degrade HSL via a lactonase mechanism. Certain strains of Variovorax and Arthrobacter utilize HSL as a sole source of nitrogen, but not as a sole source of carbon or energy. In this study, the enrichment and isolation of four strains of soil bacteria capable of utilizing HSL as a carbon and energy source are described. Phylogenetic analysis of these isolates indicates that three are distinct members of the genus Arthrobacter, whereas the fourth clusters within the non-clinical Burkholderia. The optimal pH for growth of the isolates ranged from 6.0 to 6.5, at which their HSL-dependent doubling times ranged from 1.4 to 4 h. The biodegradation of HSL by these 4 isolates far outpaced its chemical decay. HSL degradation by soil bacteria has implications for the consortial mineralization of acyl-homoserine lactones by bacteria associated with quorum sensing populations.
Keywords: Homoserine lactone degradation; Arthrobacter ; Burkholderia ; Quorum quenching
Utilization of homoserine lactone as a sole source of carbon and energy by soil Arthrobacter and Burkholderia species by Wan-Wan Yang; Jong-In Han; Jared Renton Leadbetter (pp. 47-54).
Homoserine lactone (HSL) is a ubiquitous product of metabolism. It is generated by all known biota during the editing of certain mischarged aminoacyl-tRNA reactions, and is also released as a product of quorum signal degradation by bacterial species expressing acyl-HSL acylases. Little is known about its environmental fate over long or short periods of time. The mammalian enzyme paraoxonase, which has no known homologs in bacteria, has been reported to degrade HSL via a lactonase mechanism. Certain strains of Variovorax and Arthrobacter utilize HSL as a sole source of nitrogen, but not as a sole source of carbon or energy. In this study, the enrichment and isolation of four strains of soil bacteria capable of utilizing HSL as a carbon and energy source are described. Phylogenetic analysis of these isolates indicates that three are distinct members of the genus Arthrobacter, whereas the fourth clusters within the non-clinical Burkholderia. The optimal pH for growth of the isolates ranged from 6.0 to 6.5, at which their HSL-dependent doubling times ranged from 1.4 to 4 h. The biodegradation of HSL by these 4 isolates far outpaced its chemical decay. HSL degradation by soil bacteria has implications for the consortial mineralization of acyl-homoserine lactones by bacteria associated with quorum sensing populations.
Keywords: Homoserine lactone degradation; Arthrobacter ; Burkholderia ; Quorum quenching
Characterization of the orf1glnKamtB operon of Herbaspirillum seropedicae by Lilian Noindorf; Fabiane G. M. Rego; Valter A. Baura; Rose A. Monteiro; Roseli Wassem; Leonardo M. Cruz; Liu U. Rigo; Emanuel M. Souza; Maria B. R. Steffens; Fabio O. Pedrosa; Leda S. Chubatsu (pp. 55-62).
Herbaspirillum seropedicae is an endophytic nitrogen-fixing bacterium that colonizes economically important grasses. In this organism, the amtB gene is co-transcribed with two other genes: glnK that codes for a PII-like protein and orf1 that codes for a probable periplasmatic protein of unknown function. The expression of the orf1glnKamtB operon is increased under nitrogen-limiting conditions and is dependent on NtrC. An amtB mutant failed to transport methylammonium. Post-translational control of nitrogenase was also partially impaired in this mutant, since a complete switch-off of nitrogenase after ammonium addition was not observed. This result suggests that the AmtB protein is involved in the signaling pathway for the reversible inactivation of nitrogenase in H. seropedicae.
Keywords: Herbaspirillum seropedicae ; glnK ; amtB ; Ammonium transporter; PII-like protein
Characterization of the orf1glnKamtB operon of Herbaspirillum seropedicae by Lilian Noindorf; Fabiane G. M. Rego; Valter A. Baura; Rose A. Monteiro; Roseli Wassem; Leonardo M. Cruz; Liu U. Rigo; Emanuel M. Souza; Maria B. R. Steffens; Fabio O. Pedrosa; Leda S. Chubatsu (pp. 55-62).
Herbaspirillum seropedicae is an endophytic nitrogen-fixing bacterium that colonizes economically important grasses. In this organism, the amtB gene is co-transcribed with two other genes: glnK that codes for a PII-like protein and orf1 that codes for a probable periplasmatic protein of unknown function. The expression of the orf1glnKamtB operon is increased under nitrogen-limiting conditions and is dependent on NtrC. An amtB mutant failed to transport methylammonium. Post-translational control of nitrogenase was also partially impaired in this mutant, since a complete switch-off of nitrogenase after ammonium addition was not observed. This result suggests that the AmtB protein is involved in the signaling pathway for the reversible inactivation of nitrogenase in H. seropedicae.
Keywords: Herbaspirillum seropedicae ; glnK ; amtB ; Ammonium transporter; PII-like protein
Ultrastructural organization of Alicyclobacillus tolerans strain K1T cells by Vitalii I. Duda; Natalya E. Suzina; Lyudmila O. Severina; Tatyana I. Bogdanova; Iraida A. Tsaplina; Gregorii I. Karavaiko (pp. 63-68).
Electron microscopy examinations of thin sections and freeze-fracture replicas revealed the specific ultrastructural features of Alicyclobacillus tolerans strain K1T. In particular, the cell wall displayed an ultrastructure typical of gram-positive bacteria and consisted of a thin murein layer (50–60 Å in thickness); cells exhibited a surface S-layer constituted by large hexagonally packed (p6-symmetry) rod-shaped subunits of 150–160 Å in diameter and 200 Å in height. In the cytoplasmic membrane, there were intramembrane vesicular structures that sometimes appeared as large leaflets in the central part. The cytoplasm contained numerous vesicular inclusions covered with a monolayered wall, dissimilar to bilamellar lipid membranes. Endospore coats displayed an intricate structure and consisted of three thick layers; the outer layer had an unusual fine structure; the exosporium was also found.
Keywords: Ultrastructure; Cell wall; S-layer; Membrane; Endospore coats
Ultrastructural organization of Alicyclobacillus tolerans strain K1T cells by Vitalii I. Duda; Natalya E. Suzina; Lyudmila O. Severina; Tatyana I. Bogdanova; Iraida A. Tsaplina; Gregorii I. Karavaiko (pp. 63-68).
Electron microscopy examinations of thin sections and freeze-fracture replicas revealed the specific ultrastructural features of Alicyclobacillus tolerans strain K1T. In particular, the cell wall displayed an ultrastructure typical of gram-positive bacteria and consisted of a thin murein layer (50–60 Å in thickness); cells exhibited a surface S-layer constituted by large hexagonally packed (p6-symmetry) rod-shaped subunits of 150–160 Å in diameter and 200 Å in height. In the cytoplasmic membrane, there were intramembrane vesicular structures that sometimes appeared as large leaflets in the central part. The cytoplasm contained numerous vesicular inclusions covered with a monolayered wall, dissimilar to bilamellar lipid membranes. Endospore coats displayed an intricate structure and consisted of three thick layers; the outer layer had an unusual fine structure; the exosporium was also found.
Keywords: Ultrastructure; Cell wall; S-layer; Membrane; Endospore coats
Synthesis of enzymes connected with mycoparasitism by ectomycorrhizal fungi by Joanna Mucha; Hanna Dahm; Edmund Strzelczyk; Antoni Werner (pp. 69-77).
The production of enzymes involved in mycoparasitism by several strains of ectomycorrhizal fungi: Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), S. bovinus (15-3), S. luteus (14-7) on different substrates such as colloidal chitin, mycelia of Trichoderma harzianum, T. virens and Mucor hiemalis was examined. Chitinases and β-1,3-glucanases were assayed spectrophotometrically by measuring the amount of reducing sugars releasing from suitable substrate by means of Miller’s method. β-glucosidases were determined by measuring the amount of p-nitrophenol released from p-nitrophenyl-β-D-glucopyranoside. It was observed that A. muscaria (16-3) and L. laccata (9-12) biosynthesized the highest activity of enzymes in contrast to the strains of S. bovinus and S. luteus. The mycelium of T. harzianum turned out to be the best substrate for the induction of β-1,3-glucanases and β-glucosidases for both strains of L. laccata, although the difference in the induction of chitinases in the presence of mycelia of different species of Trichoderma was not indicated.
Keywords: Chitinases; β-1,3-Glucanases; β-Glucosidases; Ectomycorrhizal fungi; Mycoparasitism
Synthesis of enzymes connected with mycoparasitism by ectomycorrhizal fungi by Joanna Mucha; Hanna Dahm; Edmund Strzelczyk; Antoni Werner (pp. 69-77).
The production of enzymes involved in mycoparasitism by several strains of ectomycorrhizal fungi: Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), S. bovinus (15-3), S. luteus (14-7) on different substrates such as colloidal chitin, mycelia of Trichoderma harzianum, T. virens and Mucor hiemalis was examined. Chitinases and β-1,3-glucanases were assayed spectrophotometrically by measuring the amount of reducing sugars releasing from suitable substrate by means of Miller’s method. β-glucosidases were determined by measuring the amount of p-nitrophenol released from p-nitrophenyl-β-D-glucopyranoside. It was observed that A. muscaria (16-3) and L. laccata (9-12) biosynthesized the highest activity of enzymes in contrast to the strains of S. bovinus and S. luteus. The mycelium of T. harzianum turned out to be the best substrate for the induction of β-1,3-glucanases and β-glucosidases for both strains of L. laccata, although the difference in the induction of chitinases in the presence of mycelia of different species of Trichoderma was not indicated.
Keywords: Chitinases; β-1,3-Glucanases; β-Glucosidases; Ectomycorrhizal fungi; Mycoparasitism
The aspartate transcarbamoylase–dihydroorotase complex in Deinococcus radiophilus has an active dihydroorotase by Donald McPhail; Margaret Shepherdson (pp. 78-81).
Aspartate transcarbamoylase (ATCase) and dihydroorotase (DHOase) catalyse the first two steps unique to pyrimidine synthesis. In many bacteria they form non-covalently bonded complexes. There are two types of DHOase, type I and type II which share a common ancestry. Type I is the more ancient form and is present in the complexes. In recently evolved bacteria the DHOase is defective and its function has been replaced by a type II DHOase which is separate from the complex. Deinococcus radiophilus diverges early on the phylogenetic tree and so might be expected to have an active type I DHOase. Purification of the 500 kDa ATCase–DHOase complex, by conventional techniques, showed it to possess an active DHOase.
Keywords: Molecular evolution; Enzyme complexes; Pyrimidine synthesis
The aspartate transcarbamoylase–dihydroorotase complex in Deinococcus radiophilus has an active dihydroorotase by Donald McPhail; Margaret Shepherdson (pp. 78-81).
Aspartate transcarbamoylase (ATCase) and dihydroorotase (DHOase) catalyse the first two steps unique to pyrimidine synthesis. In many bacteria they form non-covalently bonded complexes. There are two types of DHOase, type I and type II which share a common ancestry. Type I is the more ancient form and is present in the complexes. In recently evolved bacteria the DHOase is defective and its function has been replaced by a type II DHOase which is separate from the complex. Deinococcus radiophilus diverges early on the phylogenetic tree and so might be expected to have an active type I DHOase. Purification of the 500 kDa ATCase–DHOase complex, by conventional techniques, showed it to possess an active DHOase.
Keywords: Molecular evolution; Enzyme complexes; Pyrimidine synthesis