Deoxyribonucleoside Catabolism and Thymine Incorporation in Mutants of Escherichia coli Lacking Deoxyriboaldolase

Munch‐Petersen, Agnete

doi:10.1111/j.1432-1033.1970.tb00994.x

Cited by 38 publications

(32 citation statements)

References 27 publications

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“…Despite its wide distribution, the physiological role of the enzyme still remains obscure: nothing is known to date about its role in eucaryotes, whereas in bacterial cells, it has been proposed to function in the catabolism of deoxyribonucleosides (10,11). In Bacillus cereus, the enzyme has been reported to play a key role in the utilization of the pentose moiety of exogenous nucleosides (12,13).…”

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confidence: 99%

The First Crystal Structure of Archaeal Aldolase

Sakuraba

Tsuge

Shimoya

et al. 2003

Journal of Biological Chemistry

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A gene encoding a 2-deoxy-D-ribose-5-phosphate aldolase (DERA) homolog was identified in the hyperthermophilic Archaea Aeropyrum pernix. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized. The enzyme is an extremely thermostable DERA; its activity was not lost after incubation at 100°C for 10 min. The enzyme has a molecular mass of ϳ93 kDa and consists of four subunits with an identical molecular mass of 24 kDa. This is the first report of the presence of tetrameric DERA. The three-dimensional structure of the enzyme was determined by x-ray analysis. The subunit folds into an ␣/␤-barrel. The asymmetric unit consists of two homologous subunits, and a crystallographic 2-fold axis generates the functional tetramer. The main chain coordinate of the monomer of the A. pernix enzyme is quite similar to that of the E. coli enzyme. There was no significant difference in hydrophobic interactions and the number of ion pairs between the monomeric structures of the two enzymes. However, a significant difference in the quaternary structure was observed. The area of the subunit-subunit interface in the dimer of the A. pernix enzyme is much larger compared with the E. coli enzyme. In addition, the A. pernix enzyme is 10 amino acids longer than the E. coli enzyme in the N-terminal region and has an additional N-terminal helix. The N-terminal helix produces a unique dimer-dimer interface. This promotes the formation of a functional tetramer of the A. pernix enzyme and strengthens the hydrophobic intersubunit interactions. These structural features are considered to be responsible for the extremely high stability of the A. pernix enzyme. This is the first description of the structure of hyperthermophilic DERA and of aldolase from the Archaea domain.Aldolases catalyze carbon-carbon bond formation and cleavage and are attractive as synthetic catalysts due to their ability to produce stereospecific carbohydrates (1). They are divided into two major classes based on the mode of stabilization of reaction intermediates. Class I and II aldolases employ a Schiff base mechanism (2, 3) and a divalent metal ion for intermediate stabilization (4), respectively. 2-Deoxy-D-ribose-5-phosphate aldolase (DERA 1 ; EC 4.1.2.4) belongs to the class I aldolases and catalyzes a reversible aldol reaction between acetaldehyde and D-glyceraldehyde 3-phosphate to generate 2-deoxy-D-ribose 5-phosphate. DERA is unique in catalyzing the aldol reaction between two aldehydes as both the aldol donor and acceptor components. Its broad substrate specificity is an attractive characteristic for the production of a variety of stereospecific materials (5). The enzyme has high potential utility as an environmentally benign alternative to chiral transition metal catalysis of the asymmetric aldol reaction (6). However, the practical application of DERA is still not successful because of its low stability.Since DERA was first described by Racker (7), who reported the presence of DERA in mammalian tissue as well as in Esc...

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confidence: 99%

The First Crystal Structure of Archaeal Aldolase

Sakuraba

Tsuge

Shimoya

et al. 2003

Journal of Biological Chemistry

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“…Since a deoxyribonucleoside, when added to thc culture medium, is readily catabolized [8], the duration of thymine uptake not only will depend on the cell density but also on the concentration of the nucleoside.…”

Section: Incorporation Of Thymine Into Diva In Thymine Prototrophsmentioning

confidence: 99%

“…Two salient features of thymine-requiring mutants arc the increased catabolism of deoxynucleotides and also a low pool of dTTP and high pool levels of dUMP and dCTP [8,30].…”

Section: E Colimentioning

confidence: 99%

“…The chromatogram was developed in 0.25M lithium acetate pH 5.5-3.0M LiCl (8: 1, v/v) which allows the separation of thymine, thymidine, phosphate and deoxyribose I-phosphate. The thymine and thymidine spots were located by ultraviolet radiation, phosphate and deoxyribose 1-phosphate by spraying for phosphate [8]. Quantitative determinations were carried out as described for nucleotides.…”

Section: E N Y M C ~S W J Smentioning

confidence: 99%

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Thymine Utilization in Escherichia coli K12 on the Role of Deoxyribose 1‐Phosphate and Thymidine Phosphorylase

Jensen¹,

Leer²,

Nygaard³

1973

European Journal of Biochemistry

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Exogenously supplied thymine is only poorly utilized by wild-type cells of Escherichia coli for the synthesis of their DNA. It appears that the lack of incorporation of exogenous thymine is due to a lack of endogenous deoxyribosyl groups, which are required for the synthesis of thymidine.Data to be presented in this paper indicate that the ability of different thymine auxotrophs to grow on progressively lower thymine concentrations is a function of their capacity to increase the internal pool of deoxyribose 1 -phosphate and/or the level of thymidine phosphorylase.Thymine incorporation in wild-type cells could be observed if the cells possessed an increased deoxyribose 1 -phosphate pool.The kinetics of thymine uptake in wild-type cells, in a mutant lacking thymidine phosphorylase and in thymine auxotrophs show that the initial rates of thymine uptake are almost identical and proportional to the external thymine concentration.The experiments in vivo led us to conclude that the incorporation of exogenous thymine occurs via thymidine, which is synthesized from thymine and deoxyribose 1-phosphate, catalyzed by thymidine phosphorylase. I n accordance with this studies in vitro with purified thymidine phosphorylase showed that thymidine is synthesized from deoxyribose I-phosphate and thymine rather than through a deoxyribose transfer from a deoxynucleoside.Wild-type cells of Escherichia coli do not readily utilize exogenous thymine for the synthesis of their DNA. I n contrast, thymidine is incorporated, but the incorporation soon terminates due t o a rapid degradation of the added thymidine [l --51. Experiments with EDTA-treated cells revealed that the limited utilization of exogenous thymine cannot be ascribed solely to permeability restrictions [4,5] but is, rather, related to the availability of deoxyribosyl donors [6,7]. Thus, when deoxyribonucleosides are added to the medium, exogenous thymine is readily taken up and utilized for DNA synthesis [3-51.Abbreviations. Genes coding for: thymidylate synthetase, thy; deoxyriboaldolase, dra; phosphodeoxyribomutase, drm; purine nucleoside phosphorylase, p u p ; thymidine phosphorylase, t p p ; regulatory gene for dra, drm, tpp and pup, deoR [12].Enzymes. Deoxyriboaldolase or 2-deoxy-D-ribose-5-phosphate : acetaldehyde-lyase (EC 4.1.2.4) ; thymidine phosphorylase or thymidine : orthophosphate deoxyribosyltransferase (EC 2.4.2.4) ; purine(deoxy)ribonucleoside phosphorylase, purine-nucleoside : orthophosphate(deoxy)ribosyltransferase (EC 2.4.2.1); thymidylate synthetase or methylenetetrahydrofolate : dUMP (methyltransferase) (EC 2.1.1.); thymidine lrinase or ATP : thymidine-5'-phosphotransferase (EC 2.7.1.21).The existence of thymine-requiring mutant strains shows that exogenous thymine can be used for dTMP synthesis and that the internal deoxyribosyl groups are furnished from an increased catabolism of deoxyribonucleotides [6-S]. Thymine-requiring mutants isolated by the aminopterin procedure [7,9] require high concentrations (160-400 pM) of exogenous thymine for growth. Fro...

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“…Wild-type cells as well as mutants lacking deoxyriboaldolase have low levels of thymidine phosphorylase suggesting that such strains only to a small extent degrade deoxyribonucleotides to deoxyribose-5-phosphate [9]. This latter compound is reported to be the inducer of the deo-enzymes [16].…”

Section: Cytr Mutants With a Block I N Deoxyribonucleoside Catabolismmentioning

confidence: 99%

Mutants Constitutive for Nucleoside-Catabolizing Enzymes in Escherichia coli K 12. Isolation, Characterization and Mapping

Munch‐Petersen¹,

Nygaard²,

Hammer-Jespersen³

et al. 1972

Eur J Biochem

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Regulatory mutants of Escherichia coli, which synthesize cytidine deaminase and uridine phosphorylase constitutively have been isolated by different selection procedures and have been characterized. These mutants were also found to synthesize four other nucleoside-catabolizing enzymes, thymidine phosphorylase, deoxyriboaldolase, phosphodeoxyribomutase and purine nucleoside phosphorylase in amounts 3-to 10-fold in excess of wild-type levels.This phenotype was found to be caused by mutations in one regulatory gene designated cytll.Another class of regulatory mutants containing levels of deoxyriboaldolase, thymidine phosphorylase, phosphodeoxyribomutase and purine nucleoside phosphorylase 10-t o 100-fold above wild-type levels have been isolated by similar selection procedures. These mutants are a result of a mutation in another regulatory gene, designated deoR, and were shown to contain low, inducible levels of cytidine deaminase and uridine phosphorylase.The cytR and deoR genes have been found by P1-mediated transductions to be located a t 76.5 min and 18.5 min, respectively, on the E. coli chromosome.I n Escherichia coli, catabolism of cytidine proceeds through the reactions 1-3 in Fig.l [1,2]. The two initial reactions are catalyzed by the enzymes cytidine deaminase and uridine phosphorylase, in this paper designated the cyt-enzymes, which are present in low amounts in wild-type cells, but induced to high levels if cytidine is added to the medium [3]. Uridine docs not act as inducer in E. coli [4,5].Further catabolism of cytidine, and of deoxycytidine as well, requires the action of another group of Abbreviations and Symbols. Genes coding for: purine nucleoside phosphorylase, p p ; thymidine phosphorylase, t p p ; gene coding for phosphodeoxyribomutase, drm; gene coding for uridine phosphorylase, udp; gene coding for cytidine deaminase, cdd ; gene coding for deoxyriboaldolase, dra. Regulatory gene for: cytidine deaminase and uridine phosphorylase, cytR, for thymidine phosphorylase, deoxyriboaldolase, phosphodeoxyribomutase and purine nucleoside phosphorylase, deoR. Enzymes coded for by tpp, dra, drrn and pup, deo-enzymes; enzymes coded for by cdd and udp, cyt -enzymes.Enzymes. Purine nucleoside phosphorylase or purinenucleoside : orthophosphate (deoxy)ribosyltransferase (EC 2.4.2.1); thymidine phosphorylase or thymidine : orthophosphate deoxyribosyltransferase (EC 2.4.2.4) ; uridine phosphorylase or uridine : orthophosphate ribosyltransferase (EC 2.4.2.3) ; cytidine deaminase or (deoxy)cytidine aminohydrolase (EC 3.5.4.5); deoxyriboaldolase or %deoxy-~-ribose-5-phosphate: acetaldehyde-lyase (EC 4.1.2.4).nucleoside-catabolizing enzymes, thymidine phosphorylase, deoxyriboaldolase, phosphodeoxyribomutase (for further references see [S]). These enzymes, together with purine nucleoside phosphorylase, in the following are designated the deo-enzymes.Although the genes specifying the deo-enzymes have been shown to be closely linked and regulated by a common regulatory factor [7,8], evidence has been presented which ind...

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Deoxyribonucleoside Catabolism and Thymine Incorporation in Mutants of Escherichia coli Lacking Deoxyriboaldolase

Cited by 38 publications

References 27 publications

The First Crystal Structure of Archaeal Aldolase

The First Crystal Structure of Archaeal Aldolase

Thymine Utilization in Escherichia coli K12 on the Role of Deoxyribose 1‐Phosphate and Thymidine Phosphorylase

Mutants Constitutive for Nucleoside-Catabolizing Enzymes in Escherichia coli K 12. Isolation, Characterization and Mapping

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