Hydrolysis of oxidized nucleotides by the Escherichia coli Orf135 protein.

To examine the possibility that the Orf135 protein of Escherichia coli functions as a hydrolyzing enzyme for a damaged DNA precursor (deoxyribonucleoside 5'-triphosphate), we purified the recombinant Orf135 protein and incubated it with oxidized deoxynucleotides. Of the nucleotides tested, 2-hydroxydeoxyadenosine 5'-triphosphate, and somewhat less efficiently, 8-hydroxydeoxyguanosine 5'-triphosphate, were hydrolyzed by this protein. These damaged deoxynucleotides elicit transversion mutations in E. coli (Inoue, M., Kamiya, H., Fujikawa, K., Ootsuyama, Y., Murata-Kamiya, N., Osaki, T., Yasumoto, K., Kasai, H. (1998) J. Biol. Chem. 273, 11069-11074). These results suggest that this protein may be involved in the prevention of mutations induced by these oxidized deoxynucleotides.     

Insights into substrate recognition by the Escherichia coli Orf135 protein through its solution structure

Escherichia coli Orf135 hydrolyzes oxidatively damaged nucleotides such as 2-hydroxy-dATP, 8-oxo-dGTP and 5-hydroxy-CTP, in addition to 5-methyl-dCTP, dCTP and CTP. Nucleotide pool sanitization by Orf135 is important since nucleotides are continually subjected to potential damage by reactive oxygen species produced during respiration. Orf135 is a member of the Nudix family of proteins which hydrolyze nucleoside diphosphate derivatives. Nudix hydrolases are characterized by the presence of a conserved motif, even though they recognize various substrates and possess a variety of substrate binding pockets. We investigated the tertiary structure of Orf135 and its interaction with a 2-hydroxy-dATP analog using NMR. We report on the solution structure of Orf135, which should contribute towards a structural understanding of Orf135 and its interaction with substrates.     

Amino acid residues involved in substrate recognition of the Escherichia coli Orf135 protein

The Escherichia coli Orf135 protein, a MutT-type enzyme, hydrolyzes mutagenic 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dGTP, in addition to dCTP and 5-methyl-dCTP, and its deficiency causes increases in both the spontaneous and H(2)O(2)-induced mutation frequencies. To identify the amino acid residues that interact with these nucleotides, the Glu-33, Arg-72, Arg-77, and Asp-118 residues of Orf135, which are candidates for residues interacting with the base, were substituted, and the enzymatic activities of these mutant proteins were examined. The mutant proteins with a substitution at the 33rd, 72nd, and 118th amino acid residues displayed activities affected to various degrees for each substrate, suggesting the involvement of these residues in substrate binding. On the other hand, the mutant protein with a substitution at the 77th Arg residue had activitiy similar to that of the wild-type protein, excluding the possibility that this Arg side chain is involved in base recognition. In addition, the expression of some Orf135 mutants in orf135(-) E. coli reduced the level of formation of rpoB mutants elicited by H(2)O(2). These results reveal the residues involved in the substrate binding of the E. coli Orf135 protein.     

1H, 13C and 15N NMR assignments of the Escherichia coli Orf135 protein

Escherichia coli Orf135 protein is thought to be an enzyme that efficiently hydrolyzes oxidatively damaged nucleotides such as 2-hydroxy-dATP, 8-hydroxy-dGTP and 5-hydroxy-CTP, in addition to 5-methyl-dCTP, dCTP and CTP, thus preventing mutations in cells caused by unfavorable base pairing. Nucleotide pool sanitization by Orf135 is important since organisms are continually subjected to potential damage by reactive oxygen species produced during respiration. It is known that the frequency of spontaneous and H₂O₂-induced mutations is two to threefold higher in the orf135 ^- strain compared with the wild-type. Orf135 is a member of the Nudix family of proteins which hydrolyze nucleoside diphosphate derivatives. Nudix hydrolases are characterized by the presence of a conserved motif, although they recognize various substrates and possess a variety of substrate binding pockets. We are interested in delineating the mechanism by which Orf135 recognizes oxidatively damaged nucleotides. To this end, we are investigating the tertiary structure of Orf135 and its interaction with substrate using NMR. Herein, we report on the ¹H, ¹³C and ¹⁵N resonance assignments of Orf135, which should contribute towards a structural understanding of Orf135 and its interaction with substrate.     

Orf135 from Escherichia coli Is a Nudix hydrolase specific for CTP, dCTP, and 5-methyl-dCTP

Orf135 from Escherichia coli is a new member of the Nudix (nucleoside diphosphate linked to some other moiety, x) hydrolase family of enzymes with substrate specificity for CTP, dCTP, and 5-methyl-dCTP. The gene has been cloned for overexpression, and the protein has been overproduced, purified, and characterized. Orf135 is most active on 5-methyl-dCTP (k(cat)/K(m) = 301,000 M(-1) s(-1)), followed by CTP (k(cat)/K(m) = 47,000 M(-1) s(-1)) and dCTP (k(cat)/K(m) = 18,000 M(-1) s(-1)). Unlike other nucleoside triphosphate pyrophophohydrolases of the Nudix hydrolase family discovered thus far, Orf135 is highly specific for pyrimidine (deoxy)nucleoside triphosphates. Like other Nudix hydrolases, the enzyme cleaves its substrates to produce a nucleoside monophosphate and inorganic pyrophosphate, has an alkaline pH optimum, and requires a divalent metal cation for catalysis, with magnesium yielding optimal activity. Because of the nature of its substrate specificity, Orf135 may play a role in pyrimidine biosynthesis, lipid biosynthesis, and in controlling levels of 5-methyl-dCTP in the cell.     

Important amino acids in the phosphohydrolase module of Escherichia coli Orf135

The Escherichia coli Orf135 protein, a MutT-type enzyme, hydrolyzes 2-hydroxy-dATP and 8-hydroxy-dGTP, in addition to dCTP and 5-methyl-dCTP, and its deficiency causes increases in both the spontaneous and H(2)O(2)-induced mutation frequencies. In this study, the Gly-36, Gly-37, Lys-38, Glu-43, Arg-51, Glu-52, Leu-53, Glu-55, and Glu-56 residues of Orf135, which are conserved in the three MutT-type proteins (Orf135, MutT, and MTH1), were substituted, and the enzymatic activity of these mutant proteins was examined. The mutant proteins with a substitution at the 36th, 37th, 52nd, and 56th amino acid residues completely lost their activity. On the other hand, the mutant proteins with a substitution at the 38th, 43rd, 51st, 53rd, and 55th residues could hydrolyze 5-methyl-dCTP. Some mutants with detectable activity for 5-methyl-dCTP did not hydrolyze dCTP. Activities for known substrates (5-methyl-dCTP, dCTP, 2-hydroxy-dATP, and 8-hydroxy-dGTP) were examined in detail with the four mutants, K38R, E43A, L53A, and E55Q. These results indicate the essential residues for the activity of the Orf135 protein.     

Biotransformation of the antitumor intermediate 5-fluorouridine by recombinant Escherichia coli with high pyrimidine nucleoside phosporylase activity

Abstract

5-Fluorouridine (5-FUrd) is a precursor of the widely used antitumor drug doxifluridine. We have produced 5-FUrd by biotransformation by cloning the gene encoding pyrimidine nucleoside phosphorylase (PyNPase) from Enterobacter aero-genes CMCC (B) 45103 and expression in Escherichia coli BL21 (DE3), resulting in recombinant E. coli BL21 (DE3)/ pET28a-PyNPase. After medium optimization, the PyNPase activity in the fermentation broth was 1613 U mg^–1, which was 54-fold that of E. aerogenes. Under optimal conditions (cell concentration, 0.5 g L^–1; uridine, 10 mM; 5-fluorouracil, 45 mM; temperature, 50°C; pH, 7.8), more than 90% of uridine was converted to 5-FUrd, suggesting that this is a valuable tool for application in the preparation of antiviral and antitumor drugs.     

Human MTH1 protein hydrolyzes the oxidized ribonucleotide, 2-hydroxy-ATP

The human nucleotide pool sanitization enzyme, MTH1, hydrolyzes 2-hydroxy-dATP and 8-hydroxy-dATP in addition to 8-hydroxy-dGTP. We report here that human MTH1 is highly specific for 2-hydroxy-ATP, among the cognate ribonucleoside triphosphates. The pyrophosphatase activities for 8-hydroxy-GTP, 2-hydroxy-ATP and 8-hydroxy-ATP were measured by high-performance liquid chromatography. The kinetic parameters thus obtained indicate that the catalytic efficiencies of MTH1 are in the order of 2-hydroxy-dATP > 2-hydroxy-ATP > 8-hydroxy-dGTP > 8-hydroxy-dATP >> dGTP > 8-hydroxy-GTP > 8-hydroxy-ATP. Notably, MTH1 had the highest affinity for 2-hydroxy-ATP among the known substrates. ATP is involved in energy metabolism and signal transduction, and is a precursor in RNA synthesis. We suggest that the 2-hydroxy-ATP hydrolyzing activity of MTH1 might prevent the perturbation of these ATP-related pathways by the oxidized ATP.     

Expression of functional recombinant mussel adhesive protein Mgfp-5 in Escherichia coli

Mussel adhesive proteins have been suggested as a basis for environmentally friendly adhesives for use in aqueous conditions and in medicine. However, attempts to produce functional and economical recombinant mussel adhesive proteins (mainly foot protein type 1) in several systems have failed. Here, the cDNA coding for Mytilus galloprovincialis foot protein type 5 (Mgfp-5) was isolated for the first time. Using this cDNA, we produced a recombinant Mgfp-5 fused with a hexahistidine affinity ligand, which was expressed in a soluble form in Escherichia coli and was highly purified using affinity chromatography. The adhesive properties of purified recombinant Mgfp-5 were compared with the commercial extracted mussel adhesive Cell-Tak by investigating adhesion force using atomic force microscopy, material surface coating, and quartz crystal microbalance. Even though further macroscale assays are needed, these microscale assays showed that recombinant Mgfp-5 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments.     

Plasmid amplification in Escherichia coli after temperature upshift is impaired by induction of recombinant protein synthesis

Production of recombinant proteins often interferes with the physiology of the host organism by causing stress responses. In recombinant Escherichia coli, the cellular content of ColE1-derived plasmids and, consequently, the synthesis of the constitutively synthesized plasmid-encoded proteins generally increases after a temperature upshift. Simultaneous induction of inducible recombinant proteins that are synthesized at high levels and tend to form inclusion bodies, however, attenuates the plasmid amplification. This phenomenon was observed using temperature- as well as IPTG-inducible expression systems. Thus, high-level recombinant gene expression in connection with inclusion body formation does not only interfere with host cell but also with plasmid-related functions.     

Escherichia coli Fpg glycosylase is nonrendundant and required for the rapid global repair of oxidized purine and pyrimidine damage in vivo

Endonuclease (Endo) III and formamidopyrimidine-N-glycosylase (Fpg) are two of the predominant DNA glycosylases in Escherichia coli that remove oxidative base damage. In cell extracts and purified form, Endo III is generally more active toward oxidized pyrimidines, while Fpg is more active towards oxidized purines. However, the substrate specificities of these enzymes partially overlap in vitro. Less is known about the relative contribution of these enzymes in restoring the genomic template following oxidative damage. In this study, we examined how efficiently Endo III and Fpg repair their oxidative substrates in vivo following treatment with hydrogen peroxide. We found that Fpg was nonredundant and required to rapidly remove its substrate lesions on the chromosome. In addition, Fpg also repaired a significant portion of the lesions recognized by Endo III, suggesting that it plays a prominent role in the global repair of both purine damage and pyrimidine damage in vivo. By comparison, Endo III did not affect the repair rate of Fpg substrates and was only responsible for repairing a subset of its own substrate lesions in vivo. The absence of Endo VIII or nucleotide excision repair did not significantly affect the global repair of either Fpg or Endo III substrates in vivo. Surprisingly, replication recovered after oxidative DNA damage in all mutants examined, even when lesions persisted in the DNA, suggesting the presence of an efficient mechanism to process or overcome oxidative damage encountered during replication.     

High-level recombinant protein production by overexpression of Mlc in Escherichia coli

Escherichia coli excretes acetate during aerobic growth on LB broth containing glucose and growth ceases before depletion of glucose because of the low pH caused by the accumulation of acetate. It has been known that the acetate accumulation is reduced even when E. coli is grown in the presence of high concentration of glucose if Mlc is overexpressed. The intracellular concentration of Mlc is very low in E. coli because of autoregulation and a low efficiency of mlc translation. We constructed various mutants that can express higher levels of Mlc using site-directed mutagenesis and one of the Mlc-overproducing mutant showed reduced glucose consumption rate and low production of acetate. The mutant showed higher foreign gene expression level than that of its parental strain in the presence of glucose. These results suggest that the Mlc overproducing E. coli strain having an improved ability of glucose utilization can be a better host for high-level production of useful recombinant proteins.     

The 19-kDal protein encoded by early region 1b of adenovirus type 12 is synthesized efficiently in Escherichia coli only as a fused protein

We have constructed recombinant plasmids that direct the synthesis of the Mr 19 000 protein encoded by the adenovirus type 12 (Ad12) E1b region as either a native protein or a protein fused to the amino-terminal portion of the elongation factor EF-TuB in Escherichia coli cells. Using these recombinants, we could synthesize a large amount of the fused protein, while only a small amount of the native Mr 19 000 protein was produced. The failure to synthesize the native Mr 19 000 protein in E. coli cells was ascribed to inefficient translation.     

Control of Tn5 transposition in Escherichia coli is mediated by protein from the right repeat

The right repeat in Tn5, which encodes protein absolutely required for transposition, is also capable of inhibiting Tn5 transposition. Analysis of Tn5 mutants indicates that the left repeat is defective in supplying the transposition-inhibition function because of the sequence difference between the repeats located at nucleotide 1443; that the transposition-inhibition activity is a function of the quantity of right-repeat protein synthesis; that the smaller of the right-repeat proteins, protein 2, is sufficient for supplying the transposition-inhibition function (but not for the transposase activity); and that the transposition-inhibition function can act in trans, as opposed to the transposase activity, which functions efficiently only in cis. Gene fusion experiments indicate that the transposition-inhibition activity cannot be explained by autogenous regulation of right-repeat protein synthesis. Finally, immunoprecipitation assays of right-repeat protein-lacZ fusion proteins indicate that protein 2 is synthesized in significantly greater amounts than protein 1 in whole cells. This synthetic ratio may be important with respect to the control of Tn5 transposition.     

Extracellular accumulation of recombinant protein by Escherichia coli in a defined medium

Extracellular accumulation of recombinant proteins in the culture medium of Escherichia coli is desirable but difficult to obtain. The inner or cytoplasmic membrane and the outer membrane of E. coli are two barriers for releasing recombinant proteins expressed in the cytoplasm into the culture medium. Even if recombinant proteins have been exported into the periplasm, a space between the outer membrane and the inner membrane, the outer membrane remains the last barrier for their extracellular release. However, when E. coli was cultured in a particular defined medium, recombinant proteins exported into the periplasm could diffuse into the culture medium automatically. If a nonionic detergent, Triton X-100, was added in the medium, recombinant proteins expressed in the cytoplasm could also be released into the culture medium. It was then that extracellular accumulation of recombinant proteins could be obtained by exporting them into the periplasm or releasing them from the cytoplasm with Triton X-100 addition. The tactics described herein provided simple and valuable methods for achieving extracellular production of recombinant proteins in E. coli.     

Enhancement of recombinant protein production in Escherichia coli by coproduction of aspartase

As commonly recognized, the excretion of acetate by the aerobic growth of Escherichia coli on glucose is a manifestation of imbalanced flux between glycolysis and the tricarboxylic acid (TCA) cycle. Accordingly, this may restrict the production of recombinant proteins in E. coli, due to the limited amounts of precursor metabolites produced in TCA cycle. To approach this issue, an extra supply of intermediate metabolites in TCA cycle was made by conversion of aspartate to fumarate, a reaction mediated by the activity of L-aspartate ammonia-lyase (aspartase). As a result, in the glucose minimal medium containing aspartate, the production of two recombinant proteins, beta-galactosidase and green fluorescent protein, in the aspartase-producing strain was substantially increased by 5-fold in association with 30-40% more biomass production. This preliminary study illustrates the great promise of this approach used to enhance the production of these two recombinant proteins.     

Escherichia coli DnaK protein possesses a 5'-nucleotidase activity that is inhibited by AppppA.

AppppA and the DnaK protein have both been hypothesized to function in regulating the heat shock response of Escherichia coli. The proposals are that AppppA serves as a signal (alarmone) to turn on the heat shock response, whereas the DnaK protein is necessary to turn off the heat shock response. A simple model would be that the DnaK protein turns off the response by degrading AppppA. We disproved this model by demonstrating that the DnaK protein possesses a 5'-nucleotidase activity capable of degrading many cellular nucleotides but not AppppA. Although AppppA was not a substrate, it did inhibit the 5'-nucleotidase activity of the DnaK protein. This inhibition may be specific and have biological function since the mutant DnaK756 protein, which is defective in turning off the heat shock response, is partially desensitized to AppppA inhibition. These findings led us to consider other possible mechanisms for AppppA and the DnaK protein in heat shock regulation.     

Purification of green fluorescent protein overexpressed by a mutant recombinant Escherichia coli

Green fluorescent protein was purified from sonicated recombinant Escherichia coli and its mutant obtained after exposure to UV light. The latter overexpresses green fluorescent protein. The two-step procedure consisted of a two-phase aqueous extraction with PEG/salt and precipitation of the proteins from PEG phase by free Zn2+. The recoveries of green fluorescent protein were 73 and 83% in the cases of recombinant E. coli and its mutant, respectively. The corresponding fold purifications were 24 and 9, respectively. In both cases, the purified protein showed a single band on SDS-PAGE corresponding to 28 kDa.     

Activation of the anaerobic ribonucleotide reductase from Escherichia coli by S-adenosylmethionine.

The anaerobic ribonucleoside triphosphate reductase from Escherichia coli reduces CTP to dCTP in the presence of a second protein, named dA1, and a Chelex-treated boiled extract of the bacteria, named RT. The reaction requires S-adenosylmethionine, NADPH, dithiothreitol, ATP, and Mg2+ and K+ ions. It occurs only under anaerobic conditions. We now show that the overall reaction occurs in two steps. The first is an activation of the reductase by dA1 and RT and requires S-adenosylmethionine, NADPH, dithiothreitol, and possibly K+ ions. In the second step, the activated reductase reduces CTP to dCTP with ATP acting as an allosteric effector. During activation, S-adenosylmethionine is cleaved reductively to methionine + 5'-deoxyadenosine. This step is inhibited strongly by S-adenosylhomocysteine and various chelators. The activation of the anaerobic reductase shows a considerable similarity to that of pyruvate formate-lyase (Knappe, J., Neugebauer, F. A., Blaschkowski, H. P., and G?nzler, M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1332-1335).