Selenocysteine synthase from Escherichia coli. Nucleotide sequence of the gene (selA) and purification of the protein

The nucleotide sequence of the selA gene from Escherichia coli whose product is involved in the conversion of seryl-tRNA(Sec UCA) into selenocysteyl-tRNA(Sec UCA) was determined. selA codes for a polypeptide of a calculated Mr of 50,667; a protein of appropriate size was synthesized in vivo in a T7 promoter/polymerase system. An assay for SELA activity was devised which is based on the seryl-tRNA(Sec UCA)-dependent incorporation of [75Se] selenium into acid-insoluble material. It was used to follow SELA purification from cells that overproduced the protein from a phage T7 promoter plasmid. Purified native SELA protein migrates in gel filtration experiments with a native Mr of about 600,000. SELA contains 1 mol of bound pyridoxal 5-phosphate/mol of 50-kDa subunit. Evidence is presented that the overall conversion of seryl-tRNA(Sec UCA) to selenocysteyl-tRNA(Sec UCA) occurs at the SELA protein. SELA, therefore, has the function of a selenocysteine synthase.     

Amino acid sequence of Escherichia coli citrate synthase

Detailed evidence for the amino acid sequence of allosteric citrate synthase from Escherichia coli is presented. The evidence confirms all but 11 of the residues inferred from the sequence of the gene as reported previously [Ner, S. S., Bhayana, V., Bell, A. W., Giles, I. G., Duckworth, H. W., & Bloxham, D. P. (1983) Biochemistry 22, 5243]; no information has been obtained about 10 of these (residues 101-108 and 217-218), and we find aspartic acid rather than asparagine at position 10. Substantial regions of sequence homology are noted between the E. coli enzyme and citrate synthase from pig heart, especially near residues thought to be involved in the active site. Deletions or insertions must be assumed in a number of places in order to maximize homology. Either of two lysines, at positions 355 and 356, could be formally homologous to the trimethyllysine of pig heart enzyme, but neither of these is methylated. It appears that E. coli and pig heart citrate synthases are formed of basically similar subunits but that considerable differences exist, which must explain why the E. coli enzyme is hexameric and allosterically inhibited by NADH, while the pig heart enzyme is dimeric and insensitive to that nucleotide.     

Mechanism of the physiological reaction catalyzed by tryptophan synthase from Escherichia coli

The physiological synthesis of L-tryptophan from indoleglycerol phosphate and L-serine catalyzed by the alpha 2 beta 2 bienzyme complex of tryptophan synthase requires spatial and dynamic cooperation between the two distant alpha and beta active sites. The carbanion of the adduct of L-tryptophan to pyridoxal phosphate accumulated during the steady state of the catalyzed reaction. Moreover, it was formed transiently and without a lag in single turnovers, and glyceraldehyde 3-phosphate was released only after formation of the carbanion. These and further data prove first that the affinity for indoleglycerol phosphate and its cleavage to indole in the alpha subunit are enhanced substantially by aminoacrylate bound to the beta subunit. This indirect activation explains why the turnover number of the physiological reaction is larger than that of the indoleglycerol phosphate cleavage reaction. Second, reprotonation of nascent tryptophan carbanion is rate limiting for overall tryptophan synthesis. Third, most of the indole generated in the active site of the alpha subunit is transferred directly to the active site of the beta subunit and only insignificant amounts pass through the solvent. Comparison of the single turnover rate constants with the known elementary rate constants of the partial reactions catalyzed by the alpha and beta active sites suggests that the cleavage reaction rather than the transfer of indole or its condensation with aminoacrylate is rate limiting for the formation of nascent tryptophan.     

Substrate synergism and the steady-state kinetic reaction mechanism for EPSP synthase from Escherichia coli.

Previous studies of Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19) have suggested that the kinetic reaction mechanism for this enzyme in the forward direction is equilibrium ordered with shikimate 3-phosphate (S3P) binding first followed by phosphoenolpyruvate (PEP). Recent results from this laboratory, however, measuring direct binding of PEP and PEP analogues to free EPSPS suggest more random character to the enzyme. Steady-state kinetic and spectroscopic studies presented here indicate that E. coli EPSPS does indeed follow a random kinetic mechanism. Initial velocity studies with S3P and PEP show competitive substrate inhibition by PEP added to a normal intersecting pattern. Substrate inhibition is proposed to occur by competitive binding of PEP at the S3P site [Ki(PEP) = 6-8 mM]. To test for a productive EPSPS.PEP binary complex, the reaction order of EPSPS was evaluated with shikimic acid and PEP as substrates. The mechanism for this reaction is equilibrium ordered with PEP binding first giving a Kia value for PEP in agreement with the independently measured Kd of 0.39 mM (shikimate Km = 25 mM). Results from this study also show that the 3-phosphate moiety of S3P offers 8.7 kcal/mol in binding energy versus a hydroxyl in this position. Over 60% of this binding energy is expressed in binding of substrate to enzyme rather than toward increasing kcat. Glyphosate inhibition of shikimate turnover was poor with approximately 8 x 10(4) loss in binding capacity compared to the normal reaction, consistent with the independently measured Kd of 12 mM for the EPSPS.glyphosate binary complex. The EPSPS.glyphosate complex induces shikimate binding, however, by a factor of 7 greater than EPSPS.PEP. Carboxyallenyl phosphate and (Z)-3-fluoro-PEP were found to be strong inhibitors of the enzyme that have surprising affinity for the S3P binding domain in addition to the PEP site as measured both kinetically and by direct observation with 31P NMR. The collective data indicate that the true kinetic mechanism for EPSPS in the forward direction is random with synergistic binding occurring between substrates and inhibitors. The synergism explains how the mechanism can be random with S3P and PEP, but yet equilibrium ordered with PEP binding first for shikimate turnover. Synergism also accounts for how glyphosate can be a strong inhibitor of the normal reaction, but poor versus shikimate turnover.     

Nucleotide sequence of the Escherichia coli gene for lipid A disaccharide synthase.

The lpxB gene of Escherichia coli, believed to be the structural gene for lipid A disaccharide synthase, is located in the min 4 region of the chromosome. It is adjacent to and clockwise of the lpxA gene, which is thought to encode UDP-N-acetylglucosamine acyltransferase. Preliminary evidence suggests that lpxA and lpxB are cotranscribed in the clockwise direction and thus constitute part of a previously unknown operon (D. N. Crowell, M. S. Anderson, and C. R. H. Raetz, J. Bacteriol. 168:152-159, 1986). We now report the complete nucleotide sequence of a 1,522-base-pair PvuII-HincII fragment known to carry the lpxB gene. This sequence contained an open reading frame of 1,149 base pairs, in agreement with the predicted size, location, and orientation of lpxB. There was a second open reading frame 5' to, and in the same orientation as, lpxB that corresponded to lpxA. The ochre codon terminating lpxA was shown to overlap the methionine codon identified as the initiation codon for lpxB, suggesting that these genes are cotranscribed and translationally coupled. A third open reading frame was also shown to begin at the 3' end of lpxB with analogous overlap between the opal codon terminating lpxB and the methionine codon that putatively initiates translation downstream of lpxB in the clockwise direction. These results argue that at least three genes constitute a translationally coupled operon in the min 4 region of the E. coli chromosome. The accompanying paper by Tomasiewicz and McHenry (J. Bacteriol. 169:5735-5744, 1987) presents 4.35 kilobases of DNA sequence, beginning at the 3' end of lpxB, and argues that dnaE and several other open reading frames may be members of this operon.     

Nucleotide sequence of the tag gene from Escherichia coli.

We have determined the complete nucleotide sequence of the tag gene, encoding 3-methyladenine DNA glycosylase I from Escherichia coli. From the nucleotide sequence it is deduced that the tag enzyme consists of 187 amino-acids and has a calculated molecular weight of 21.1 kdaltons. The tag enzyme is unusually rich in cysteine (8 residues) with a cluster of three consecutive cysteines near the C-terminal end. The tag coded DNA glycosylase does not show significant sequence homology to the alkA coded glycosylase in spite of that both of these enzymes catalyze the release of free 3-methyladenine from alkylated DNA.     

Analysis of the metal requirement of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

The three isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli were overproduced, purified, and characterized with respect to their requirement for metal cofactor. The isolated isozymes contained 0.2-0.3 mol of iron/mol of enzyme monomer, variable amounts of zinc, and traces of copper. Enzymatic activity of the native enzymes was stimulated 3-4-fold by the addition of Fe2+ ions to the reaction mixture and was eliminated by treatment of the enzymes with EDTA. The chelated enzymes were reactivated by a variety of divalent metal ions, including Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mn2+, Ni2+, and Zn2+. The specific activities of the reactivated enzymes varied widely with the different metals as follows: Mn2+ greater than Cd2+, Fe2+ greater than Co2+ greater than Ni2+, Cu2+, Zn2+ much greater than Ca2+. Steady state kinetic analysis of the Mn2+, Fe2+, Co2+, and Zn2+ forms of the phenylalanine-sensitive isozyme (DAHPS(Phe)) revealed that metal variation significantly affected the apparent affinity for the substrate, erythrose 4-phosphate, but not for the second substrate, phosphoenolpyruvate, or for the feedback inhibitor, L-phenylalanine. The tetrameric DAHPS(Phe) exhibited positive homotropic cooperativity with respect to erythrose 4-phosphate, phophoenolpyruvate, and phenylalanine in the presence of all metals tested.     

Substrate-induced membrane association of phosphatidylserine synthase from Escherichia coli.

To better establish the intracellular location of the phosphatidylserine synthase of Escherichia coli and hence better understand how it is regulated in the cell, we compared the size, function, and binding properties of the enzyme made in vitro with the enzyme found in cell lysates and with the purified enzyme. The enzyme made either in vivo or in an active form in vitro was found primarily associated with the ribosomal fraction of the cell and had the same apparent molecular mass as the purified enzyme. These results were unaffected by the presence of protease inhibitors. Addition of unsupplemented E. coli membranes or membranes supplemented with phosphatidylethanolamine did not affect the subcellular distribution of the enzyme in these experiments. However, addition of membranes supplemented with either the lipid substrate, CDP-diacylglycerol, or the lipid product, phosphatidylserine, resulted in membrane association by the enzyme rather than ribosomal association. Addition of membranes supplemented with acidic lipids also brought about membrane association, but this association was primarily ionic since it was disrupted by high salt concentrations. These results strongly suggest that the ribosomal location of this enzyme is not the result of some modification event occurring after cell lysis and that the normal functioning of the enzyme involves membrane association which is primarily induced by the presence of a membrane-associated substrate.     

The crystal structure of methylglyoxal synthase from Escherichia coli.

BACKGROUND: The reaction mechanism of methylglyoxal synthase (MGS) is believed to be similar to that of triosephosphate isomerase (TIM). Both enzymes utilise dihydroxyacetone phosphate (DHAP) to form an enediol(ate) phosphate intermediate as the first step of their reaction pathways. However, the second catalytic step in the MGS reaction pathway is characterized by the elimination of phosphate and collapse of the enediol(ate) to form methylglyoxal instead of reprotonation to form the isomer glyceraldehyde 3-phosphate. RESULTS: The crystal structure of MGS bound to formate and substoichiometric amounts of phosphate in the space group P6522 has been determined at 1.9 A resolution. This structure shows that the enzyme is a homohexamer composed of interacting five-stranded beta/alpha proteins, rather than the hallmark alpha/beta barrel structure of TIM. The conserved residues His19, Asp71, and His98 in each of the three monomers in the asymmetric unit bind to a formate ion that is present in the crystallization conditions. Differences in the three monomers in the asymmetric unit are localized at the mouth of the active site and can be ascribed to the presence or absence of a bound phosphate ion. CONCLUSIONS: In agreement with site-directed mutagenesis and mechanistic enzymology, the structure suggests that Asp71 acts as the catalytic base. Further, Asp20 and Asp101 are involved in intersubunit salt bridges. These salt bridges may provide a pathway for transmitting allosteric information.     

Identification and sequence of the drpA gene from Escherichia coli.

The drpA gene of Escherichia coli encodes a factor that is involved in global RNA synthesis. We establish that the drpA gene has been successfully cloned and describe the fine-structure map of three drpA-(Ts) mutations as well as the complete nucleotide sequence of the drpA gene. We identified a major sigma-70 promoter for the drpA gene on the bases of (i) its similarity to the consensus sequence and (ii) S1 protection and primer extension mapping data. In addition, the nucleotide sequence revealed a pair of dnaA boxes and a factor-independent terminator at the 5' end and 3' end of the gene, respectively. The deduced amino acid sequence of the DrpA protein showed a nucleotide-binding pocket found in some ATPases.     

The nucleotide sequence of the ilvBN operon of Escherichia coli: sequence homologies of the acetohydroxy acid synthase isozymes.

Three acetohydroxy acid synthase isozymes, AHAS I (ilvBN), AHAS II (ilvGM) and AHAS III (ilvIH) catalyze the first step of the parallel isoleucine-valine biosynthetic pathway in Escherichia coli. Previous DNA sequence and protein purification data have shown that AHAS II and AHAS III are composed of large and small subunits encoded in the ilvGMEDA and ilvIH operons, respectively. Recent protein purification and characterization data have demonstrated that the AHAS I isozyme is also composed of large and small subunits (L. Eoyang, L. and P. M. Silverman [1984] J. Bacteriol. 157:184-189). Now the complete DNA sequence of the operon encoding the AHAS I isozyme has been determined. These data show that both AHAS I subunits (Mr 60,400 and Mr 11,100) are encoded in this operon. The coordinant regulation of both genes of the ilvBN operon has also been demonstrated. Comparisons of the DNA sequences of the genes encoding all three AHAS isozymes have been performed. Conserved homologies were observed between both the large and small subunits of all three isozymes. The closest homology was seen between the AHAS I and AHAS II isozymes. On the basis of these comparisons a rationale for the evolution of the AHAS isozymes in E. coli has been proposed.     

The nucleotide sequence of asparagine tRNA from Escherichia coli.

The nucleotide seuquence of Escherichia coli asparagine tRNA was determined to be pU-C-C-U-C-U-G-s4U-A-G-U-U-C-A-G-D-C-G-G-D-A-G-A-A-C-G-G-C-G-G-A-C-U-Q-U-U-t6A-A-phi-C-C-G-U-A-U-m G-U-C-A-C-U-G-G-T-phi-C-G-A-G-U-C-C-A-G-U-C-A-G-A-G-G-A-G-C-C-AOH. Its D-stem and D-loop have almost the same sequence as Escherichia coli aspartate tRNA.     

The catalytic mechanism of tryptophan synthase from Escherichia coli. Kinetics of the reaction of indole with the enzyme--L-serine complexes

The mechanism by which indole condenses with L-serine in the active site of tryptophan synthase was studied by the stopped-flow technique. The single turnover occurs by rapid binding of indole to the pre-formed enzyme--L-serine complex, followed by C--C bond formation, reprotonation of the alpha carbon carbanion of L-tryptophan, and its final release. The effects of isotopic substitution at C-3 of indole, of pH, and of the presence of indolepropanol phosphate on these processes were also studied. The mechanism of binding of indole complements the known mechanisms of binding of L-serine and L-tryptophan to give a detailed picture of the mechanism of catalysis. It invokes two competent species of enzyme--L-serine complexes, leading to a branched pathway for the central condensation process. The rates of dehydration of L-serine and reprotonation of the carbanion of L-tryptophan are probably limited by rearrangements at the active site. Analysis of absorption, fluorescence and circular dichroic spectra, as well as of published data on the stereoisomers obtained by reduction with borohydride, suggests that the rearrangement includes a reorientation of the pyridoxal phosphate C-4' atom. The mechanism provides a detailed framework for explaining all available information, including the activating effect of the alpha subunit on the reaction catalyzed by the beta 2 subunit.     

Vibrationally enhanced hydrogen tunneling in the Escherichia coli thymidylate synthase catalyzed reaction

The enzyme thymidylate synthase (TS) catalyzes a complex reaction that involves forming and breaking at least six covalent bonds. The physical nature of the hydride transfer step in this complex reaction cascade has been studied by means of isotope effects and their temperature dependence. Competitive kinetic isotope effects (KIEs) on the second-order rate constant (V/K) were measured over a temperature range of 5-45 degrees C. The observed H/T ((T)V/K(H)) and D/T ((T)V/K(D)) KIEs were used to calculate the intrinsic KIEs throughout the temperature range. The Swain-Schaad relationships between the H/T and D/T V/K KIEs revealed that the hydride transfer step is the rate-determining step at the physiological temperature of Escherichia coli (20-30 degrees C) but is only partly rate-determining at elevated and reduced temperatures. H/D KIE on the first-order rate constant k(cat) ((D)k = 3.72) has been previously reported [Spencer et al. (1997) Biochemistry 36, 4212-4222]. Additionally, the Swain-Schaad relationships between that (D)k and the V/K KIEs reported here suggested that at 20 degrees C the hydride transfer step is the rate-determining step for both rate constants. Intrinsic KIEs were calculated here and were found to be virtually temperature independent (DeltaE(a) = 0 within experimental error). The isotope effects on the preexponential Arrhenius factors for the intrinsic KIEs were A(H)/A(T) = 6.8 +/- 2.8 and A(D)/A(T) = 1.9 +/- 0.25. Both effects are significantly above the semiclassical (no-tunneling) predicted values and indicate a contribution of quantum mechanical tunneling to this hydride transfer reaction. Tunneling correction to transition state theory would predict that these isotope effects on activation parameters result from no energy of activation for all isotopes. Yet, initial velocity measurements over the same temperature range indicate cofactor inhibition and result in significant activation energy on k(cat) (4.0 +/- 0.1 kcal/mol). Taken together, the temperature-independent KIEs, the large isotope effects on the preexponential Arrhenius factors, and a significant energy of activation all suggest vibrationally enhanced hydride tunneling in the TS-catalyzed reaction.     

Characterization of a specific interaction between Escherichia coli thymidylate synthase and Escherichia coli thymidylate synthase mRNA.

Previous studies have shown that human TS mRNA translation is controlled by a negative autoregulatory mechanism. In this study, an RNA electrophoretic gel mobility shift assay confirmed a direct interaction between Escherichia coli (E.coli) TS protein and its own E.coli TS mRNA. Two cis-acting sequences in the E.coli TS mRNA protein-coding region were identified, with one site corresponding to nucleotides 207-460 and the second site corresponding to nucleotides 461-807. Each of these mRNA sequences bind TS with a relative affinity similar to that of the full-length E.coli TS mRNA sequence (IC50 = 1 nM). A third binding site was identified, corresponding to nucleotides 808-1015, although its relative affinity for TS (IC50 = 5.1 nM) was lower than that of the other two cis-acting elements. E.coli TS proteins with mutations in amino acids located within the nucleotide-binding region retained the ability to bind RNA while proteins with mutations at either the nucleotide active site cysteine (C146S) or at amino acids located within the folate-binding region were unable to bind TS mRNA. These studies suggest that the regions on E.coli TS defined by the folate-binding site and/or critical cysteine sulfhydryl groups may represent important RNA binding domains. Further evidence is presented which demonstrates that the direct interaction with TS results in in vitro repression of E.coli TS mRNA translation.     

Escherichia coli cyclopropane fatty acid synthase.

Escherichia coli fatty acid cyclopropane synthase (CFAS) was overproduced and purified as a His6-tagged protein. This recombinant enzyme is as active as the native enzyme with a Km of 90 microm for S-AdoMet and a specific activity of 5 x 10(-2) micromol.min(-1).mg(-1). The enzyme is devoid of organic or metal cofactors and is unable to catalyze the wash-out of the methyl protons of S-AdoMet to the solvent, data that do not support the ylide mechanism. Inactivation of the enzyme by 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), a pseudo first-order process with a rate constant of 1.2 m(-1).s(-1), is not protected by substrates. Graphical analysis of the inactivation by DTNB revealed that only one cysteine is responsible for the inactivation of the enzyme. The three strictly conserved Cys residues among cyclopropane synthases, C139, C176 and C354 of the E. coli enzyme, were mutated to serine. The relative catalytic efficiency of the mutants were 16% for C139S, 150% for C176S and 63% for C354S. The three mutants were inactivated by DTNB at a rate comparable to the rate of inactivation of the His6-tagged wild-type enzyme, indicating that the Cys responsible for the loss of activity is not one of the conserved residues. Therefore, none of the conserved Cys residues is essential for catalysis and cannot be involved in covalent catalysis or general base catalysis. The inactivation is probably the result of steric hindrance, a phenomenon irrelevant to catalysis. It is very likely that E. coli CFAS operates via a carbocation mechanism, but the base and nucleophile remain to be identified.     

Purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli.

A procedure for the purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli is described. Homogeneous enzyme of specific activity 17.7 units/mg was obtained in 22% yield. The key purification step involves substrate elution of the enzyme from a cellulose phosphate column. The subunit Mr was estimated to be 49 000 by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The native Mr was estimated to be 55 000 by gel filtration, indicating that the enzyme is monomeric.