Direct expression of active spinach glycolate oxidase in Escherichia coli.

Spinach glycolate oxidase (GAO) was expressed in Escherichia coli using the T7 RNA polymerase promotor. The enzyme accounts for approx. 1% of the soluble protein fraction and is expressed as a soluble and active enzyme. Comparison with GAO expressed in Saccharomyces cerevisiae (Macheroux, P., Massey, V., Thiele, D.J. and Volokita, M. (1991) Biochemistry 30, 4612-4619) showed that the GAO expressed in E. coli has identical physico-chemical features to the wild-type enzyme, but is expressed at a level approx. 15-fold higher than in the yeast system.

Evidence for two conformational states of thioredoxin reductase from Escherichia coli: use of intrinsic and extrinsic quenchers of flavin fluorescence as probes to observe domain rotation.

Thioredoxin reductase (TrxR) from Escherichia coli consists of two globular domains connected by a two-stranded beta sheet: an FAD domain and a pyridine nucleotide binding domain. The latter domain contains the redox-active disulfide composed of Cys 135 and Cys 138. TrxR is proposed to undergo a conformational change whereby the two domains rotate 66 degrees relative to each other (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800-816), placing either redox active disulfide (FO conformation) or the NADPH binding site (FR conformation) adjacent to the flavin. This domain rotation model was investigated by using a Cys 138 Ser active-site mutant. The flavin fluorescence of this mutant is only 7% that of wild-type TrxR, presumably due to the proximity of Ser 138 to the flavin in the FO conformation. Reaction of the remaining active-site thiol, Cys 135, with phenylmercuric acetate (PMA) causes a 9.5-fold increase in fluorescence. Titration of the PMA-treated mutant with the nonreducing NADP(H) analogue, 3-aminopyridine adenine dinucleotide phosphate (AADP+), results in significant quenching of the flavin fluorescence, which demonstrates binding adjacent to the FAD, as predicted for the FR conformation. Wild-type TrxR, with or without PMA treatment, shows similar quenching by AADP+, indicating that it exists mostly in the FR conformer. These findings, along with increased EndoGluC protease susceptibility of PMA-treated enzymes, agree with the model that the FO and FR conformations are in equilibrium. PMA treatment, because of steric limitations of the phenylmercuric adduct in the FO form, forces the equilibrium to the FR conformer, where AADP+ binding can cause fluorescence quenching and conformational restriction favors proteolytic susceptibility.

Formation and properties of mixed disulfides between thioredoxin reductase from Escherichia coli and thioredoxin: evidence that cysteine-138 functions to initiate dithiol-disulfide interchange and to accept the reducing equivalent from reduced flavin.

Mutation of one of the cysteine residues in the redox active disulfide of thioredoxin reductase from Escherichia coli results in C135S with Cys138 remaining or C138S with Cys135 remaining. The expression system for the genes encoding thioredoxin reductase, wild-type enzyme, C135S, and C138S has been re-engineered to allow for greater yields of protein. Wild-type enzyme and C135S were found to be as previously reported, whereas discrepancies were detected in the characteristics of C138S. It was shown that the original C138S was a heterogeneous mixture containing C138S and wild-type enzyme and that enzyme obtained from the new expression system is the correct species. C138S obtained from the new expression system having 0.1% activity and 7% flavin fluorescence of wild-type enzyme was used in this study. Reductive titrations show that, as expected, only 1 mol of sodium dithionite/mol of FAD is required to reduce C138S. The remaining thiol in C135S and C138S has been reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) to form mixed disulfides. The half time of the reaction was <5 s for Cys138 in C135S and approximately 300 s for Cys135 in C138S showing that Cys138 is much more reactive. The resulting mixed disulfides have been reacted with Cys32 in C35S mutant thioredoxin to form stable, covalent adducts C138S-C35S and C135S-C35S. The half times show that Cys138 is approximately fourfold more susceptible to attack by the nucleophile. These results suggest that Cys138 may be the thiol initiating dithiol-disulfide interchange between thioredoxin reductase and thioredoxin.

Exploring the conformational equilibrium of E. coli thioredoxin reductase: characterization of two catalytically important states by ultrafast flavin fluorescence spectroscopy.

The conformational dynamics of wild-type Escherichia coli thioredoxin reductase (TrxR) and the mutant enzyme C138S were studied by ultrafast time-resolved fluorescence of the flavin cofactor in combination with circular dichroism (both in the flavin fingerprint and far-UV regions) and steady-state fluorescence and absorption spectroscopy. The spectroscopic data show two conformational states of the enzyme (named FO and FR), of which the physical characteristics differ considerably. Ultrafast fluorescence lifetime measurements make it possible to distinguish between the two different populations: Dominant picosecond lifetimes of approximately 1 ps (contribution 75%) and 7 ps (8%) are associated with the FO species in TrxR C138S. Long-lived fluorescence with two time constants in the range of 0.2-1 ns (total contribution 17%) originates from enzyme molecules in the FR conformation. The near absence of fast lifetime components in oxidized wild-type TrxR supports the idea of this enzyme being predominantly in the FR conformation. The emission spectrum of the FO conformation is blue-shifted with respect to that of the FR conformation. Because of the large difference in fluorescence characteristics, fluorescence measurements on time scales longer than 100 ps are fully determined by the fraction of enzyme molecules in the FR conformation. Binding of the thiol reagent phenyl mercuric acetate to wild-type enzyme and TrxR C138S stabilizes the enzymes in the FR conformation. Specific binding of the NADPH-analog, AADP(+), to the FR conformation resulted in dynamic fluorescence quenching in support of the multiple quenching sites model. Raising the temperature from 277K-323K resulted in a moderate shift to the FR conformation for TrxR C138S. High concentrations of the cosolvent glycerol triggered the domain rotation from the FO to the FR conformation.

Application of a single-plasmid vector for mutagenesis and high-level expression of thioredoxin reductase and its use to examine flavin cofactor incorporation.

Thioredoxin reductase from Escherichia coli is a dimeric enzyme containing one FAD and one redox-active disulfide per monomer and catalyzes the transfer of electrons from NADPH to thioredoxin, which subsequently performs several important cellular functions. To overcome problems with site-directed mutagenesis and low expression, the thioredoxin reductase gene was adapted for use in the plasmid vector pSL350 (Brosius, J., Methods Enzymol. 216, 469-483, 1992), which is designed both for protein expression and for production of single-stranded template DNA for mutagenesis, and examined expression of wild-type thioredoxin reductase under different growth conditions. In the absence of IPTG inducer, expression of thioredoxin reductase in saturated cultures accounts for 19% of the soluble protein, and with 1 mM IPTG expression increases to 61%. Some of the thioredoxin reductase is expressed as apoenzyme with the amount of apoenzyme increasing at higher IPTG concentrations, accounting for as high as 68% of the total thioredoxin reductase expressed. The apoenzyme in cell extracts is activated rapidly by addition of FAD, indicating correct folding of the enzyme in the absence of cofactor. Purification of wild-type thioredoxin reductase from the new system yielded 189 mg of enzyme from a 300-ml uninduced culture. The new plasmid was also used to generate an N155Y mutant which is purified and partially characterized.

Sequence of the Klebsiella aerogenes urease genes and evidence for accessory proteins facilitating nickel incorporation.

A 4.8-kilobase-pair region of cloned DNA encoding the genes of the Klebsiella aerogenes urease operon has been sequenced. Six closely spaced open reading frames were found: ureA (encoding a peptide of 11.1 kilodaltons [kDa]), ureB (11.7-kDa peptide), ureC (60.3-kDa peptide), ureE (17.6-kDa peptide), ureF (25.2-kDa peptide), and ureG (21.9-kDa peptide). Immediately after the ureG gene is a putative rho-dependent transcription terminator. The three subunits of the nickel-containing enzyme are encoded by ureA, ureB, and ureC based on protein structural studies and sequence homology to jack bean urease. Potential roles for ureE, ureF, and ureG were explored by deleting these accessory genes from the operon. The deletion mutant produced inactive urease, which was partially purified and found to have the same subunit stoichiometry and native size as the active enzyme but which contained no significant levels of nickel. The three accessory genes were able to activate apo-urease in vivo when they were cloned into a compatible expression vector and cotransformed into cells carrying the plasmid containing ureA, ureB, and ureC. Thus, one or more of the ureE, ureF, or ureG gene products are involved in nickel incorporation into urease.

Potential active-site base of thioredoxin reductase from Escherichia coli: examination of histidine245 and aspartate139 by site-directed mutagenesis.

It has been proposed that an acid-base catalyst facilitates the reduction of thioredoxin by thioredoxin reductase from Escherichia coli [O'Donnell, M. E., & Williams, C. H. Jr. (1983) J. Biol. Chem. 252, 13795-13805]. The X-ray crystal structure reveals two groups which could potentially fulfill this role: His245 and Asp139. Using site-directed mutagenesis, His245 was changed to asparagine (H245N) and alanine (H245A) and Asp139 was changed to glutamate (D139E), asparagine (D139N), and leucine (D139L). Steady-state kinetic analysis of the His245 mutants gave turnover numbers and Km values similar to those of wild-type thioredoxin reductase. All three Asp139 mutants were altered in their overall kinetic properties: D139E had 38% of wild-type activity, D139N had 1.5%, and D139L had no measureable activity. Rate constants for the NADPH to 3-acetylpyridine adenine dinucleotide phosphate transhydrogenase activity were similar for all of the Asp139 and His245 mutants and wild-type thioredoxin reductase. Stopped-flow kinetic measurements of the reductase half-reaction of H245A and H245N gave rate constants that were up to 2-fold faster than those found for wild-type thioredoxin reductase, while all of the Asp139 mutants had rate constants comparable to those of wild-type. To further examine the causes of the low overall activity of D139N, the oxidative half-reaction was measured. The reoxidation of reduced D139N mixed with oxidized thioredoxin occurred at a very slow rate constant of 0.23 s-1-about 1% that of wild-type enzyme. We suggest that Asp139 is the active-site acid catalyst which functions to protonate the thiolate anion of reduced thioredoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

High-level expression in Escherichia coli and purification of the membrane-bound form of cytochrome b(5).

Expression of the membrane-bound form of rabbit cytochrome b(5) in Escherichia coli has been significantly improved through the use of the T7 expression vector pLW01 (A. Bridges, L. Gruenke, Y.-T. Chang, I. Vakser, G. Loew, and L. Waskell, 1998, J. Biol. Chem. 273, 17036-17049) in conjunction with strain C41(DE3) (B. Miroux and J. Walker, 1996, J. Mol. Biol. 260, 289-298). Cell cultures expressing the cytochrome b(5) contained an average of 820 mg/liter of culture and reached peak levels as high as 1100 mg/liter when higher antibiotic concentrations were used. Maximal levels were obtained from cultures when expression was induced with 10 microM IPTG. Approximately 90% of the cytochrome b(5) was expressed as apoprotein which was reconstituted by addition of exogenous heme. The cytochrome b(5) was purified from detergent-solubilized bacterial membranes using anion-exchange chromatography on DEAE-Sepharose followed by size-exclusion chromatography on Superdex-75. Purification of cytochrome b(5) from a 500-ml culture yielded 121 mg of protein which had a specific content of 50 nmol of heme per milligram of protein with an overall recovery of 35%. The final cytochrome b(5) was free of any detectable contaminants when analyzed by SDS-PAGE.

Purification, characterization, and in vivo reconstitution of Klebsiella aerogenes urease apoenzyme.

Urease was purified from recombinant Klebsiella aerogenes which was grown in the absence of nickel. The protein was inactive and contained no transition metals, yet it possessed the same heteropolymeric structure as native enzyme, demonstrating that Ni is not required for intersubunit association. Ni did, however, substantially increase the stability of the intact metalloprotein (Tm = 79 degrees C) compared with apoenzyme (Tm = 62 degrees C), as revealed by differential scanning calorimetric analysis. An increased number of histidine residues were accessible to diethyl pyrocarbonate in apourease compared with holoenzyme, consistent with possible Ni ligation by histidinyl residues. Addition of Ni to purified apourease did not yield active enzyme; however, urease apoenzyme was very slowly activated in vivo by addition of Ni ions to Ni-free cell cultures, even after treatment of the cells with spectinomycin to inhibit protein synthesis. In contrast, sonicated cells and cells treated with dinitrophenol or dicyclohexylcarbodiimide were incapable of activating apourease. These results indicate that apourease activation is an energy-dependent process that is destroyed by cell disruption.

Regulation of gene expression and cellular localization of cloned Klebsiella aerogenes (K. pneumoniae) urease.

The genes for Klebsiella aerogenes (K. pneumoniae) urease were cloned and the protein was overexpressed (up to 18% of total protein consisted of this enzyme) in several hosts. The small size of the DNA encoding urease (3.5 kb), the restriction map, and the regulation of enzyme expression directed by the recombinant plasmid are distinct from other cloned ureases. Nickel concentration did not affect urease gene expression, as demonstrated by the high levels of apoenzyme measured in cells grown in nickel-free media. However, nickel was required for urease activity. The overproducing recombinant strain was used for immunogold electron microscopic localization studies to demonstrate that urease is a cytoplasmic enzyme.

Purification, characterization, and genetic organization of recombinant Providencia stuartii urease expressed by Escherichia coli.

Recombinant urease from Providencia stuartii has been expressed in and purified from Escherichia coli, and the genetic organization of the structural genes has been determined. Urease expression was induced by urea and repressed by nitrogen-rich components in the medium. The urease protein was purified 331-fold by DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and phenyl-Superose chromatographies with a 7.3% yield. The enzyme possessed a Km for urea of 9.3 mM and hydrolyzed urea at a Vmax of 7,100 mumol/min per mg. P. stuartii urease is composed of three polypeptides (Mrs, 73,000, 10,0000, and 9,000) denoted by alpha, beta, and gamma. The native enzyme is best described as (alpha 1 beta 2 gamma 2)2, based on a native Mr of 230,000, obtained by gel filtration chromatography, and on the Coomassie blue staining intensities of the individual subunits. Atomic absorption analysis of the pure protein revealed 1.9 +/- 0.1 nickel ions per alpha 1 beta 2 gamma 2 unit. In vitro transcription-translation analysis of transposon insertion mutants of the recombinant urease demonstrated that the urease peptides are encoded on adjacent DNA sequences and transcribed as a polycistronic mRNA in the order gamma, beta, and then alpha. Three urease-defective insertion mutants were identified that did not affect synthesis of urease subunit polypeptides, indicating that some nickel processing, enzyme activation, or other function may also be necessary for producing an active urease.

Klebsiella aerogenes urease gene cluster: sequence of ureD and demonstration that four accessory genes (ureD, ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis.

The region located immediately upstream from the Klebsiella aerogenes urease structural genes was sequenced and shown to possess an open reading frame capable of encoding a 29.8-kDa peptide. Deletions were generated in this gene, denoted ureD, and in each of the genes (ureE, ureF, and ureG) located immediately downstream of the three structural genes. Transformation of the mutated plasmids into Escherichia coli resulted in high levels of urease expression, but the enzyme was inactive (deletions in ureD, ureF, or ureG) or only partially active (deletions in ureE). Ureases were purified from the recombinant cells and shown to be identical to control enzyme when analyzed by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis; however, in every case the activity levels correlated to nickel contents as analyzed by atomic absorption analysis. UreD, UreE, UreF, and UreG peptides were tentatively identified by gel electrophoretic comparison of mutant and control cell extracts, by in vivo expression of separately cloned genes, or by in vitro transcription-translation analyses; the assignments were confirmed for UreE and UreG by amino-terminal sequencing. The latter peptides (apparent M(r)s, 23,900 and 28,500) were present at high levels comparable to those of the urease subunits, whereas the amounts of UreF (apparent M(r), 27,000) and UreD (apparent M(r), 29,300) were greatly reduced, perhaps because of the lack of good ribosome binding sites in the regions upstream of these open reading frames. These results demonstrate that all four accessory genes are necessary for the functional incorporation of the urease metallocenter.

Crystal structure of Klebsiella aerogenes UreE, a nickel-binding metallochaperone for urease activation.

UreE is proposed to be a metallochaperone that delivers nickel ions to urease during activation of this bacterial virulence factor. Wild-type Klebsiella aerogenes UreE binds approximately six nickel ions per homodimer, whereas H144*UreE (a functional C-terminal truncated variant) was previously reported to bind two. We determined the structure of H144*UreE by multi-wavelength anomalous diffraction and refined it to 1.5 A resolution. The present structure reveals an Hsp40-like peptide-binding domain, an Atx1-like metal-binding domain, and a flexible C terminus. Three metal-binding sites per dimer, defined by structural analysis of Cu-H144*UreE, are on the opposite face of the Atx1-like domain than observed in the copper metallochaperone. One metal bridges the two subunits via the pair of His-96 residues, whereas the other two sites involve metal coordination by His-110 and His-112 within each subunit. In contrast to the copper metallochaperone mechanism involving thiol ligand exchanges between structurally similar chaperones and target proteins, we propose that the Hsp40-like module interacts with urease apoprotein and/or other urease accessory proteins, while the Atx1-like domain delivers histidyl-bound nickel to the urease active site.

Preparation of figure 8 and cruciform DNAs and their use in studies of the kinetics of branch migration.

We have re-examined the kinetics of the branch migration of double-stranded DNA that is mediated by the stepwise movement of the Holliday junction. This work revises and extends our previous treatment (Thompson, B. J., Camien, M. N., and Warner, R. C. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2299-2303). New methodology and new highly purified substrates have been used. The latter include figure 8s prepared from phage G4 DNA by annealing single-stranded components and two sizes of a novel cruciform. We treat the process as a one-dimensional diffusion based on the random walk, the mathematical basis of which is discussed in detail. The step rate is shown to be 3 orders of magnitude slower than we reported previously. The most important contribution to the erroneously high rate was a result of the presence of EDTA in the spreading solution used for electron microscopy at that time. A second contribution of about 4-fold resulted from catalysis by EcoRI and other proteins. The rates reported here are for the uncatalyzed reaction.