The PLP-dependent biotin synthase from Escherichia coli: mechanistic studies

Biotin synthase (BioB), an iron-sulfur enzyme, catalyzes the last step of the biotin biosynthesis pathway. The reaction consists in the introduction of a sulfur atom into two non-activated C-H bonds of dethiobiotin. Substrate radical activation is initiated by the reductive cleavage of S-adenosylmethionine (AdoMet) into a 5'-deoxyadenosyl radical. The recently described pyridoxal 5'-phosphate-bound enzyme was used to show that only one molecule of AdoMet, and not two, is required for the formation of one molecule of biotin. Furthermore 5'-deoxyadenosine, a product of the reaction, strongly inhibited biotin formation, an observation that may explain why BioB is not able to make more than one turnover. However this enzyme inactivation is not irreversible.     

Escherichia coli 987P pilus: purification and partial characterization

The Escherichia coli somatic pilus, 987P, has been purified after removal by homogenization from a 987P+ enterotoxigenic E. coli. Cell-free pili were precipitated by the addition of MgCl2, collected, and dissolved in MgCl2-free buffer. Five cycles of precipitation and dissolving resulted in a preparation of 987P that was judged to be homogeneous based on electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the electron microscope, 987P was rod shaped, having a diameter of 7 nm and an apparent axial hole. Cells and membrane vesicles were not observed in the purified pilus preparation. Electrophoresis of 987P through sodium dodecyl sulfate-polyacrylamide gels resulted in a single band when the sample was denatured in the absence of mercaptoethanol and in two bands when the sample was denatured in the presence of mercaptoethanol. The calculated molecular weight of 987 was variable, depending upon the polyacrylamide concentration and whether mercaptoethanol was included in the denaturing solution. Chemically, 987P is composed primarily of protein but also contains an unidentified amino sugar. The amino terminal amino acid of 987P is alanine and its isoelectric point is pH 3.7. 987P possesses no detectable hemagglutinating activity.     

The purification and properties of Escherichia coli methylglyoxal synthase

1. Methylglyoxal synthase was purified over 1500-fold from glycerol-grown Escherichia coli K 12 strain CA 244. The purified enzyme was inactivated by heat or proteolysis, had a molecular weight of approx. 67000, a pH optimum of 7.5 and was specific for dihydroxyacetone phosphate with K(m) 0.47mm. 2. The possibility that a Schiff-base intermediate was involved in the reaction mechanism was investigated but not confirmed. 3. The purified enzyme lost activity, especially at low temperature, but could be stabilized by P(i). Two binding sites for P(i) may be present on the enzyme. Of other compounds tested only the substrate, dihydroxyacetone phosphate, and bovine serum albumin showed any significant stabilizing effect. 4. Phosphoenolpyruvate, 3-phosphoglycerate, PP(i) and P(i) were potent inhibitors of the enzyme. Kinetic experiments showed that PP(i) was apparently a simple competitive inhibitor, but inhibition by the other compounds was more complex. In the presence of P(i) the enzyme behaved co-operatively, with at least three binding sites for dihydroxyacetone phosphate. 5. It is proposed that methylglyoxal synthase and glyceraldehyde 3-phosphate dehydrogenase play important roles in the catabolism of the triose phosphates in E. coli. Channelling of dihydroxyacetone phosphate via methylglyoxal would not be linked to ATP formation and could be involved in the uncoupling of catabolism and anabolism.     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli: positive cooperativity with substrates and inhibitors

Investigations have been made at pH 6.0 of the effect of chorismate and adamantane derivatives on the mutase and dehydrogenase activities of hydroxyphenylpyruvate synthase from Escherichia coli. When used over a wide range of concentrations, chorismate 5,6-epoxide, chorismate 5,6-diol, adamantane-1,3-diacetate, adamantane-1-acetate, adamantane-1-carboxylate, and adamantane-1-phosphonate give rise to nonlinear plots of the reciprocal of the initial velocity of each reaction as a function of the inhibitor concentration. The inhibitors do not induce the enzyme to undergo polymerization and have only a small effect on the S20,w value of the enzyme as determined by using sucrose density gradient centrifugation. At low substrate concentration, low concentrations of adamantane-1-acetate cause activation of both the mutase and dehydrogenase activities while at higher concentrations this compound functions as an inhibitor. When chorismate and prephenate are varied over a wide range of concentrations, double-reciprocal plots of the data indicate that the reactions exhibit positive cooperativity. The addition of albumin eliminates the cooperative interactions associated with substrates but has little effect on those associated with inhibitors.     

Acetohydroxy acid synthase I of Escherichia coli: purification and properties.

Several properties of the three acetohydroxy acid synthases of Escherichia coli have been compared in crude extracts. The three enzymes can be readily distinguished from each other. Acetohydroxy acid synthase I, the product of the ilvB gene, has been purified to near homogeneity. The purification was made possible by the fact that the enzyme was maintained in buffers of a high ionic strength or in buffers containing glycerol. Density gradient centrifugation studies indicated that the enzyme exists as a dimer of subunits of similar (60,000) molecular weight in buffers containing glycerol with or without two of the cofactors. Mg2+ and thiamine diphosphate. When flavine adenine dinucleotide was added along with Mg2+ and thiamine diphosphate, an increase in the rate of sedimentation occurred that was thought to be due to a rapid tetramer-dimer interconversion. The addition of pyruvate, the substrate, along with the three cofactors, resulted in a further increase in sedimentation rate, due presumably to an increase in the tetramer-to-dimer ratio. The addition of valine to the complete system resulted in maintenance of the enzyme in the dimeric state concomitant with inhibition of enzyme activity.     

Derepression of anthranilate synthase in purified minicells of Escherichia coli containing the Col-trp plasmid

Purified minicells of Escherichia coli K-12 containing the plasmid Col-trp(+) or Col-trpA2 could be derepressed for the synthesis of anthranilate synthase, the first enzyme encoded in the trp operon. Non-plasmid-containing, deoxyribonucleic acid-deficient minicells could not be derepressed. Derepressed enzyme synthesis was initiated by l-tryptophan starvation. The kinetics of derepression were studied with minicells containing the Col-trpA2 plasmid. The derepression curves were biphasic with a rapid initial rate of enzyme synthesis followed by a slower rate of synthesis. The presence of l-tryptophan (20 to 50 mug/ml) or chloramphenicol (200 mug/ml) abolished enzyme synthesis. The presence of rifamycin SV (280 mug/ml) partially inhibited enzyme synthesis after at least 3.5 min of exposure. The ratio of minicell-to-cell synthetic capacity was 1:2.4 when compared on the basis of derepressed enzyme activity per unit cell volume. This work demonstrates that plasmid-containing minicells are capable of considerable functional protein and messenger ribonucleic acid synthesis and that the regulation of at least the trp operon is similar in minicells to that observed in cells.     

Amplification and substantial purification of cardiolipin synthase of Escherichia coli.

A simple, specific, and sensitive assay procedure for cardiolipin synthase of Escherichia coli has been developed. This measures the radioactivity of glycerol formed from phosphatidyl [2-3H]glycerol and is mainly based on the findings that 400 mM phosphate and 0.015% Triton X-100 markedly activate the enzyme. Cardiolipin synthase was amplified 760-fold upon induction with isopropyl beta-D-thiogalactoside in cells harboring a pBR322 derivative in which the cls gene encoding this enzyme was preceded by the tac promoter. Under these conditions, cardiolipin content increased, membrane potential decreased, spheroplasts became fragile, cells lost viability, and inducer-resistant mutants appeared at a high frequency. The amplification enabled the isolation of an enzyme preparation with a specific activity approximately 10,000-times higher than that of wild-type whole cell lysate. This purification was simply achieved by extraction of the crude membrane fraction with Triton X-100 and a single phosphocellulose column chromatography. This preparation, together with the crude envelope fraction, was used to characterize the basic properties of E. coli cardiolipin synthase, some of which were utilized in setting up the assay conditions.     

The cloning and over-expression of PABA synthase in E. coli

Both the genes encoding E. coli p-aminobenzoic acid synthase have been cloned and an overproducing strain has been obtained. The partial purification of the large subunit is described. The kinetic properties of the cloned enzyme, while similar to those reported for the B. subtilis enzyme, show some differences to those reported for the S. griseus enzyme.     

Chorismate mutase/prephenate dehydrogenase from Escherichia coli K12: purification, characterization, and identification of a reactive cysteine

The bifunctional enzyme involved in tyrosine biosynthesis, chorismate mutase/prephenate dehydrogenase, has been isolated from extracts of a regulatory mutant of Escherichia coli K12. The pure enzyme is a homodimer of total molecular weight 78 000 and displays Michaelis-Menten kinetics for both activities. Fingerprinting and amino acid sequencing of tryptic and thermolytic peptides of the S-[14C]carboxymethylated enzyme allowed the identification of three unique cysteine-containing sequences per subunit. Chemical modification of the native enzyme with 5,5'-dithiobis(2-nitrobenzoate) or iodoacetamide showed that one sulfhydryl group per subunit was particularly reactive, and the integrity of this group was essential for both enzymic activities. This work supports previous proposals for a close spatial relationship between the active sites.     

Structure and regulation of the anthranilate synthase genes in Pseudomonas aeruginosa: II. Cloning and expression in Escherichia coli

The genes for the large and small subunits of anthranilate synthase (trpE and trpG, respectively) have been cloned from Pseudomonas aeruginosa PAC174 into E. coli by R-prime formation with the broad-host- range plasmid R68.44. Sequential subcloning into plasmid vectors reduced the active Pseudomonas DNA fragment to a length of 3.1 kb. We obtained evidence that this region contains the promoter for its own expression and retains a vestigial regulatory response to tryptophan scarcity or excess.      

Purification and characterization of selenomethionyl thymidylate synthase from Escherichia coli: comparison with the wild-type enzyme

Replacement of methionine (Met) residues by selenomethionine (SeMet) was recently shown to facilitate the crystallographic analysis of protein structure through the application of multi-wavelength anomalous diffraction techniques [Yang et al. (1990) Science (Washington, D.C.) 249, 1398-1405]. The availability of SeMet-containing proteins provides an excellent opportunity to evaluate the effects of the complete replacement of Met by SeMet. We chose to compare the properties of selenomethionyl thymidylate synthase isolated from Escherichia coli DL41 (a methionine auxotroph) and wild-type (wt) enzyme obtained from E. coli Rue10. An improved purification procedure for thymidylate synthase was developed which permitted the isolation of 25 mg of pure protein from 2 g of E. coli in 90% yield in no more than 8 h. The pure wt and SeMet enzymes exhibited specific activities 40% higher than published values. Thermal stability studies at 30 degrees C in degassed buffer showed that the SeMet enzyme (t1/2 67 h) was 8-fold less stable than wt enzyme (t1/2 557 h). The half-lives for the latter enzymes in nondegassed buffers at 30 degrees C were decreased by 2-fold, thus indicating the sensitivity of the enzyme to dissolved oxygen. Both enzymes exhibited essentially the same kinetic and binding properties, including Km(dUMP) (1.2 x 10(-6) M), specificity constant (1.6 x 10(6) s-1 M-1), and Kd for 5-fluorodeoxyuridylate binding (1.2 nM) in covalent inhibitory ternary complexes. In addition, X-ray crystallographic analysis by difference Fourier synthesis showed there was no significant difference in conformation between the SeMet enzyme and the wt enzyme.     

Expression and purification of His-tagged flavonol synthase of Camellia sinensis from Escherichia coli

Flavonols, a class of bioactive polyphenols present in plants, are the products of flavonol desaturation catalyzed by flavonol synthase (FLS). We cloned the cDNA coding for the enzyme FLS from Camellia sinensis (CsFLS) by end-to-end PCR followed by 5'- and 3'-RACE. The putative CsFLS had 333 amino acid residues, displayed identities to the FLSs of Arabidopsis and Ginkgo of 53% and 52.5%, respectively, and contained several conserved elements found in the 2-oxoglutarate-Fe(II)-dioxygenase superfamily. The cDNA of CsFLS was subcloned into pET28a(+) and introduced into Escherichia coli (BL21-CodonPlus-RIL). Induction with 0.1mM IPTG at low temperature (20 degrees C) led to higher amounts of CsFLS in the soluble fraction than induction at 30 degrees C. The enzyme aggregated into inclusion bodies could be rescued by denaturation with 6M urea and purification with a His.Bind purification kit. The purified protein was desalted by Amicon Ultra-15 centrifugal filter unit, and the His-tag was removed with thrombin. The finally purified protein was assayed with dihydroquercetin as substrate and the products were analyzed by HPLC. The addition of FeSO(4) to the buffers used in the CsFLS purification significantly increased the recovery of active enzyme. The CsFLS obtained in this study was found to have higher specific activity and lower K(m) than previously reported FLSs.     

Crystal structure of Escherichia coli malate synthase G complexed with magnesium and glyoxylate at 2.0 A resolution: mechanistic implications

The crystal structure of selenomethionine-substituted malate synthase G, an 81 kDa monomeric enzyme from Escherichia coli has been determined by MAD phasing, model building, and crystallographic refinement to a resolution of 2.0 A. The crystallographic R factor is 0.177 for 49 242 reflections observed at the incident wavelength of 1.008 A, and the model stereochemistry is satisfactory. The basic fold of the enzyme is that of a beta8/alpha8 (TIM) barrel. The barrel is centrally located, with an N-terminal alpha-helical domain flanking one side. An inserted beta-sheet domain folds against the opposite side of the barrel, and an alpha-helical C-terminal domain forms a plug which caps the active site. Malate synthase catalyzes the condensation of glyoxylate and acetyl-coenzyme A and hydrolysis of the intermediate to yield malate and coenzyme A, requiring Mg(2+). The structure reveals an enzyme-substrate complex with glyoxylate and Mg(2+) which coordinates the aldehyde and carboxylate functions of the substrate. Two strictly conserved residues, Asp631 and Arg338, are proposed to provide concerted acid-base chemistry for the generation of the enol(ate) intermediate of acetyl-coenzyme A, while main-chain hydrogen bonds and bound Mg(2+) polarize glyoxylate in preparation for nucleophilic attack. The catalytic strategy of malate synthase appears to be essentially the same as that of citrate synthase, with the electrophile activated for nucleophilic attack by nearby positive charges and hydrogen bonds, while concerted acid-base catalysis accomplishes the abstraction of a proton from the methyl group of acetyl-coenzyme A. An active site aspartate is, however, the only common feature of these two enzymes, and the active sites of these enzymes are produced by quite different protein folds. Interesting similarities in the overall folds and modes of substrate recognition are discussed in comparisons of malate synthase with pyruvate kinase and pyruvate phosphate dikinase.     

GroE-dependent expression and purification of pig heart mitochondrial citrate synthase in Escherichia coli

Citrate synthase (CS) is a dimeric, mitochondrial protein, composed of two identical subunits (M(r) 48969 each). The nuclear-encoded alpha-helical protein is imported into mitochondria post-translationally where it catalyses the first step of the citric cycle. Furthermore, the pathway of thermal unfolding as well as the folding pathway was studied extensively, making CS a well-suited substrate protein for studying chaperone function. In chaperone research the quality of the substrate proteins is essential to guaranty the reproducibility of the results. In this context, we here describe the GroE-enhanced recombinant expression and purification of CS. CS was expressed in E. coli by using an arabinose regulated T7 promotor. Under standard expression conditions only insoluble, inactive CS was detected. Interestingly, the expression of soluble and active CS was possible when GroEL/GroES was co-expressed. Furthermore, a shift to lower expression temperatures increased the amount of soluble, active CS. We describe for the first time, the purification of CS in soluble and active form by following a CiPP strategy (capture, intermediate purification, polishing). After the initial capturing step on DEAE-Sephacel the protein was further purified on a Q-Sepharose column. After these two steps of anion-exchange chromatography a final size-exclusion chromatography step on a Superdex 75-pg column yields CS with a purity over 99%. Using this expression and purification strategy 1 mg CS per g E. coli wet weight were purified.     

Chorismate synthase of Neurospora crassa: a flavoprotein

Chorismate synthase is purified from Neurospora crassa. The final step is accomplished by preparative electrophoresis. Its purity is estimated at ≥90% on the basis of analytical polyacrylamide gel electrophoresis. The enzyme appears to be active in at least two multimeric states, with a subunit molecular weight of ~55,000. The purified enzyme preparation is absolutely dependent on the presence of a reducing system, which can readily be provided under aerobic conditions by NADPH plus FMN or under stringent anaerobic conditions by dithionite. The following evidence implicates a physiological role for FMN in N. crassa chorismate synthase activity: (a) a preferential stimulation of activity by NADPH and FMN over other pyridine and flavin nucleotides, respectively, in both impure and purified enzyme preparations; (b) an alteration of the Chromatographic pattern of the enzyme on diethylaminoethylcellulose by the addition of FMN to the elution buffer; (c) an apparent binding of FMN to the enzyme as exhibited by gel filtration in the presence of the substrate, 3-enolpyruvylshikimate 5-phosphate; (d) a requirement for preliminary incubation with FMN, in concert with the substrate, to eliminate a reaction lag (i.e., to activate the enzyme); (e) a substrate-dependent diaphorase activity exhibited by purified enzyme preparations in the presence of FMN and NADPH. The observed activation and alteration of Chromatographic behavior of chorismate synthase by FMN suggest that the flavin nucleotide influences the conformation of the enzyme. The ability to replace NADPH and FMN with dithionite suggests that FMN mediates the flow of electrons from a source of reducing power (NADPH) to some enzymic site important to the function of the enzyme. Hence, the diaphorase activity which is observed as intrinsic to chorismate synthase of N. crassa may be significant from the standpoint of catalysis or may have importance as a regulatory mechanism.