One-step, highly efficient site-directed mutagenesis by toxic protein selection.

A fast and efficient site-directed mutagenesis method has been developed, using the newly constructed plasmid pTPS19, which expresses the toxic CcdB protein originally encoded by the E. coli F plasmid. Once the target gene is cloned into pTPS19, desired mutations can be introduced with two primers. The first contains the desired mutation, and the second is designed to create a +1 frame shift in the ccdB gene to inactivate the CcdB protein. The mutants can be directly selected on LB plates containing IPTG, through which the toxic CcdB protein is induced, thereby eliminating cells carrying wild-type parental plasmids. Based on stringent selection through the toxic CcdB protein, mutagenesis efficiency of 90%-100% was reached even after one round of transformation.

The structure and domain organization of Escherichia coli isocitrate lyase.

Enzymes of the glyoxylate-bypass pathway are potential targets for the control of many human diseases caused by such pathogens as Mycobacteria and Leishmania. Isocitrate lyase catalyses the first committed step in this pathway and the structure of this tetrameric enzyme from Escherichia coli has been determined at 2.1 A resolution. E. coli isocitrate lyase, like the enzyme from other prokaryotes, is located in the cytoplasm, whereas in plants, protozoa, algae and fungi this enzyme is found localized in glyoxysomes. Comparison of the structure of the prokaryotic isocitrate lyase with that from the eukaryote Aspergillus nidulans reveals a different domain structure following the deletion of approximately 100 residues from the larger eukaryotic enzyme. Despite this, the active sites of the prokaryotic and eukaryotic enzymes are very closely related, including the apparent disorder of two equivalent segments of the protein that are known to be involved in a conformational change as part of the enzyme's catalytic cycle.

A study of conserved in-loop and out-of-loop glycine residues in the large subunit of ribulose bisphosphate carboxylase/oxygenase by directed mutagenesis.

The replacement of all 22 completely conserved glycine residues in the large subunit of ribulose bisphosphate carboxylase/oxygenase from Anacystis nidulans by directed mutagenesis is described. In each beta/alpha barrel of the large subunit there are 12 completely conserved glycines in six of eight loops at the C-termini of eight beta-strands and four in loops at N-terminal ends of the beta-strands. Two completely conserved glycines are also in each beta/alpha barrel backbone and four more are in a large N-terminal portion preceding the barrel in a given L subunit. Substitution of glycines in loops that are C-terminal to beta-strands by proline was more deleterious to carboxylase activity than that by alanine supporting the postulates that these loops contribute to catalysis and substrate binding and that in some cases the glycines may serve as hinges enabling movement of the loops. In contrast, substitution of glycines at the N-terminal ends of beta-strands in the beta/alpha barrel more often led to failure to detect L subunits or their assembly into L8S8 complex. Substitution of these and the other conserved glycines by proline was more deleterious to carboxylase activity than by alanine in enzymes that assembled.

Lysine 194 is functional in isocitrate lyase from Escherichia coli.

Lysine 194 in conserved stretch 1 of tetrameric isocitrate lyase from Escherichia coli has been replaced by using the restriction-enzyme-site elimination method of directed mutagenesis. Expression of subunits of each variant and of wild-type (wt) enzyme was equivalent and all variants assembled into tetrameric proteins. The variants K194R and K194H had kcat values relative to that of wt enzyme taken as 100 of 11 and 7, respectively. Km values for Mg2+-Ds-isocitrate (in mM units) were: 0.13 for wt-enzyme; 0.12 for the K194R variant; and 0.55 for the K194H variant. Substitution at position 194 of Leu or Glu resulted in zero catalytic activity. These results establish that Lys 194 is another functional residue in conserved stretch one of isocitrate lyase from E. coli besides H184, K193, C195, and H197. Because K194 can be specifically replaced by the basic residues His and Arg with resultant lowered activity and by His with an increased Km value, it may contribute to a cation center and facilitate substrate binding as well as orientation of the developing transition state.

Activity and carboxylation specificity factor of mutant ribulose 1,5-bisphosphate carboxylase/oxygenase from Anacystis nidulans.

The values of molecular carboxylase activity kcat and carboxylation specificity factor tau for mutant ribulose 1,5-bisphosphate carboxylase (rubisco) from Anacystis nidulans decreased as compared to those of the wild type recombinant rubisco. The substitution of five amino acid residues in rubisco large subunit Lys,Ala,Ser,Thr,Leu(339-343)Phe,Leu,Met,Ile,Lys had kcat decreased by 90% and tau by 36.3%. The same parameters for mutants with the single replacements decreased: for Thr342Ile kcat by 40.5% and tau by 16.7%, and for mutant Leu343Lys kcat by 48.1% and tau by 18.5%. Mutant rubisco with three amino acid residues changed Val,Asp,Leu(346-348)Tyr,His,Thr was inactive. The substitution Leu326Ile decreased kcat by 54.4% and tau by 34.2%; and change Ser328Ala decreased kcat only by 5.6% but tau by 41.5%. Replacement Asn123His decreased kcat by 16.5%. Significance of the non conservative amino acid residues for carboxylase activity and ribulose-1,5-bisphosphate partition is discussed.

Serine319 and 321 are functional in isocitrate lyase from Escherichia coli.

With site-directed mutagenesis, Ser319 and Ser321 in conserved stretch 3 of tetrameric isocitrate lyase from Escherichiacoli were each substituted with alanine, cysteine, asparagine, or threonine in addition to simultaneous alanine/alanine substitutions. Besides their absolute conservation in all aligned isocitrate lyase sequences, the location of these serine residues, which flank a completely conserved proline, had been suggested in the active site in previous research by studies of photoinactivation of the enzyme by vanadate [Ko et al. (1992) J Biol Chem 267:91]. All substitutions for Ser321 and 319 except by threonine appreciably reduced the kcat of E. coli isocitrate lyase relative to that for wild-type (100) as follows: S319A, 0.4; S319C, 0.05; S319N, 0.01; S319T, 32.3; S321A, 2.9; S321C, 0.3; S321N, 0.1; S321T, 0.3; and S319A/S321A, 0, with little or no effect on the Km for the substrate Mg2+-Ds-isocitrate. The most active variant S319T exhibited threefold less activity than the wild-type enzyme; all variants assembled into tetramers. The S319T mutant isocitrate lyase was 100-fold more active than the S321T variant. This observation suggests that the requirement for a beta-hydroxymethyl group of serine in catalysis is less important at position 319 than at position 321. Although most singly substituted variants had very low isocitrate lyase activity, all variants harboring mutant isocitrate lyase of very low activity did grow on acetate asa sole carbon source albeit with longer doubling times and lag phases. Substitution of Pro320 by Ala, Asp, Gly, or His was highly detrimental to activity and increased the Km for substrate 3.5- to 8-fold; this suggests that Pro fixes the location of adjacent Ser OH groups and facilitates substrate binding and catalysis. From these collective results, it is proposed that Ser319 and Ser321 are involved in E.coli isocitrate lyase catalysis, perhaps by stabilizing the postulated reaction intermediate succinate trianion in the aci-carboxylate form and the related transition state via hydrogen bonding.

Sequence analysis of plasmid pCC5.2 from cyanobacterium Synechocystis PCC 6803 that replicates by a rolling circle mechanism.

The cyanobacterium Synechocystis sp. strain PCC 6803 contains several cryptic plasmids of 2.4, 5.2, and about 50 or 100 kbp. The complete nucleotide sequence of the 5.2-kbp plasmid, pCC5.2, has been analyzed and is reported here. This plasmid contains 5214 bp and 53.1% A+T. Six open reading frames, ORFs A-F (encoding peptides of larger than 90 amino acid residues), were located on both strands of pCC5.2. ORF B codes for a potential replication protein containing 971 amino acids, for which there are three homologous proteins encoded by other cyanobacterial plasmids. ORF C encodes a polypeptide of 93 amino acids which shares homologies with products of two ORFs found in the protein database. Counterparts of the products of ORF A, D, E, and F could not be found in the protein database. Detection of a single-stranded DNA intermediate during replication of pCC5.2 indicates that this plasmid may also replicate by a rolling circle mechanism, as has been reported for pCA2.4 and pCB2.4 from the same strain of Synechocystis (PCC 6803).

Cysteine 195 has a critical functional role in catalysis by isocitrate lyase from Escherichia coli.

Cysteine 195 in isocitrate lyase from Escherichia coli has been replaced by directed mutagenesis. Substitution by Ser yields enzyme with a k(cat) that is 0.03% that of wild type, and substitution by Ala, Gly, Thr, or Val yields completely inactive enzyme. The present results are consistent with a functional role of Cys 195.

The consequences of replacing histidine 356 in isocitrate lyase from Escherichia coli.

Isocitrate lyase from Escherichia coli has been expressed in transformed E. coli JE10 cells lacking the isocitrate lyase (icl) gene. After directed mutagenesis of icl by the restriction-site elimination method, partially purified isocitrate lyase mutants in which His 356 has been converted to Lys, Arg, Gln, Asp, or Leu have been characterized after induction of transformed, induced JE10 cells. Values of kcat compared to those for wild-type (wt) enzyme (100) at 37 degrees C, pH 7.3, are 18, 1, <1, 0, and 0 for H356K, H356R, H356E, H356Q, and H356L mutant enzymes, respectively. Km values for the 1:1 Mg-isocitrate complex (in millimolar units) are: 0.13, wt; 0.11, H356K; and 0.63, H356R. Further chromatographic purification of isocitrate lyase yields highly purified wt, H356K, and H356R enzymes. The pH profile of the stability of isocitrate lyase, which has never been reported, showed that the H356R enzyme was unstable in the pH range investigated; the wt and H356R variant differed but each was sufficiently stable to study the pH dependence of catalysis. The log kcat/pH profiles for highly purified wt and H356K enzymes are roughly bell-shaped and have pKa and pKb values for dissociation of an ionizable group on the enzyme-substrate complex of <6.3 and 8.4 for wt and 5.9 and 7.9 for H356K enzymes. Plots of pKm vs pH were different for the wt and H356K variant. Values of pKa and pKb (derived from log kcat/Km plots vs pH) for the dissociation of an activity-related ionizable group on the variant were 5.3 and 7.6, whereas the analogous pKb value for the wt enzyme was 8.4. The data suggest that His 356 is an important functional residue in isocitrate lyase, perhaps in deprotonating isocitrate during catalytic cleavage.

A sensitive, simultaneous analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase efficiencies: Graphical determination of the CO2/O 2 specificity factor.

A simple approach to determine CO2/O2 specificity factor (τ) of ribulose 1,5-bisphosphate carboxylase/oxygenase is described. The assay measures the amount of CO2 fixation at varying [CO2]/[O2] ratios after complete consumption of ribulose 1,5-bisphosphate (RuBP). Carbon dioxide fixation catalyzed by the carboxylase was monitored by directly measuring the moles of (14)CO2 incorporated into 3-phosphoglycerate (PGA). This measurement at different [CO2]/[O2] ratios is used to determine graphically by several different linear plots the total RuBP consumed by the two activities and the CO2/O2 specificity factor. The assay can be used to measure the amounts of products of the carboxylase and oxygenase reactions and to determine the concentration of the substrate RuBP converted to an endpoint amount of PGA and phosphoglycolate. The assay was found to be suitable for all [CO2]/[O2] ratios examined, ranging from 14 to 215 micromolar CO2 (provided as 1-16 mM NaHCO3) and 614 micromolar O2 provided as 50% O2. The procedure described is extremely rapid and sensitive. Specificity factors for enzymes of highly divergent τ values are in good agreement with previously published data.

The complete DNA sequence and replication analysis of the plasmid pCB2.4 from the cyanobacterium Synechocystis PCC 6803.

Both strands of small plasmid pCB2.4 from the unicellular cyanobacterium Synechocystis PCC 6803 have been sequenced and analyzed. pCB2.4 contains 2345 bp and 57% A+T. Three open reading frames (ORFs) were identified in the nucleotide sequence of one strand of pCB2.4. ORF1 and ORF3 may encode basic proteins with calculated pI values of 7.71 and 9.96, respectively. The products of these ORFs have been compared to published protein sequences in databases and do not show significant homology to other proteins including replication proteins. pCB2.4 is not similar at the global DNA level to another 2.4-kbp plasmid, pCA2.4, present in the same cells, which has been found to replicate by a rolling-circle mechanism via a single-stranded intermediate. However, a short region (37 bp) is similar in pCA2.4 and pCB2.4. This region contains a potential nicking site (GATA) for replication proteins encoded by a group of plasmids that replicate by a rolling-circle mechanism and might be used as the origin of replication for these plasmids. Detection of a low level of single-stranded intermediate for pCB2.4 in the Synechocystis 6803 suggests that pCB2.4 may also replicate by a rolling-circle mechanism.

The importance of four histidine residues in isocitrate lyase from Escherichia coli.

By site-directed mutagenesis, substitutions were made for His-184 (H-184), H-197, H-266, and H-306 in Escherichia coli isocitrate lyase. Of these changes, only mutations of H-184 and H-197 appreciably reduced enzyme activity. Mutation of H-184 to Lys, Arg, or Leu resulted in an inactive isocitrate lyase, and mutation of H-184 to Gln resulted in an enzyme with 0.28% activity. Nondenaturing polyacrylamide gel electrophoresis demonstrated that isocitrate lyase containing the Lys, Arg, Gln, and Leu substitutions at H-184 was assembled poorly into the tetrameric subunit complex. Mutation of H-197 to Lys, Arg, Leu, and Gln resulted in an assembled enzyme with less than 0.25% wild-type activity. Five substitutions for H-266 (Asp, Glu, Val, Ser, and Lys), four substitutions for H-306 (Asp, Glu, Val, and Ser), and a variant in which both H-266 and H-306 were substituted for showed little or no effect on enzyme activity. All the H-197, H-266, and H-306 mutants supported the growth of isocitrate lyase-deficient E. coli JE10 on acetate as the sole carbon source; however, the H-184 mutants did not.

Active-site histidines in recombinant cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase examined by site-directed mutagenesis.

The functions of His(291), His(295) and His(324) at the active-site of recombinant A. nidulans ribulose-1,5-bisphosphate carboxylase/ oxygenase have been explored by site-directed mutagenesis. Replacement of His(291) by K or R resulted in unassembled proteins, while its replacement by E, Q or N resulted in assembled but inactive proteins. These results are in accord with a metal ion-binding role of this residue in the activated ternary complex by analogy to x-ray crystallographic analyses of tobacco and spinach enzymes.His(324) (H327 in spinach), which is located within bonding distance of the 5-phosphate of bound bi-substrate analog 2-carboxyarabinitol 1,5-bisphosphate in the crystal structures, has been substituted by A, K, R, Q and N. Again with the exception of the H324K and R variants, these changes resulted in detectable assembled protein. The mutant H324A protein exhibited no detectable carboxylase activity, whereas the H324Q and H324N changes resulted in purifiable holoenzyme with 2.0 and 0.1% of the recombinant wild-type specific carboxylase activity, respectively. These results are consistent with a phosphate binding role for this residue.The replacement of His(295), which has been suggested to aid in phosphate binding, with Ala in the A. nidulans enzyme leads to a mutant with 5.8% of the recombinant wild-type carboxylase activity. All other mutations at this position resulted in unassembled proteins. Purified H295A and H324Q enzymes had elevated Km(RuBP) values and unchanged CO2/O2 specificity factors compared to recombinant wild-type.

A small plasmid, pCA2.4, from the cyanobacterium Synechocystis sp. strain PCC 6803 encodes a rep protein and replicates by a rolling circle mechanism.

Different cryptic plasmids are widely distributed in many strains of cyanobacteria. A small cryptic plasmid, pCA2.4, from Synechocystis strain PCC 6803 was completely sequenced, and its replication mode was determined. pCA2.4 contained 2,378 bp and encoded a replication (Rep) protein, designated RepA. An analysis of the deduced amino acid sequence revealed that RepA of pCA2.4 has significant homology with Rep proteins of pKYM from Shigella sonnei, a pUB110 plasmid family from gram-positive bacteria, and with a protein corresponding to an open reading frame in a Nostoc plasmid and open reading frame C of Plectonema plasmid pRF1. pKYM and pUB110 family plasmids replicate by a rolling circle mechanism in which a Rep protein nicks the origin of replication to allow the generation of a single-stranded plasmid as a replication intermediate. RepA encoded by pC2.4 was expressed in Escherichia coli cells harboring a vector, pCRP336, containing the entire repA gene. The observed molecular weight of RepA was consistent with the value of 39,200 calculated from its deduced amino acid sequence, as was the N-terminal sequence analysis done through the 12th residue. Single-stranded plasmid DNA of pCA2.4 that was specifically degraded by S1 nuclease was detected in Synechocystis cells by Southern hybridization. These observations suggest that pCA2.4 replicates by a rolling circle mechanism in Synechocystis cells.

Leucine 332 influences the CO2/O2 specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase from Anacystis nidulans.

The role of Leu 332 in ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans was investigated by site-directed mutagenesis. Substitutions of this residue with Met, Ile, Val, Thr, or Ala decreased the CO2/O2 specificity factor by as much as 67% and 96% for the Ile mutant in the presence of Mg2+ and Mn2+, respectively. For the Met, Ile, and Ala mutants in the presence of Mg2+, no loss of oxygenase activity was observed despite the loss of greater than 65% of the carboxylase activity relative to the wild-type enzyme. In the presence of Mn2+, carboxylase activities for mutant enzymes were reduced to approximately the same degree as was observed in the presence of Mg2+, although oxygenase activities were also reduced to similar extents as carboxylase activities. Only minor changes in Km(RuBP) were observed for all mutants in the presence of Mg2+ relative to the wild-type enzyme, indicating that Leu 332 does not function in RuBP binding. These results suggest that in the presence of Mg2+, Leu 332 contributes to the stabilization of the transition state for the carboxylase reaction, and demonstrate that it is possible to affect only one of the activities of this bifunctional enzyme.

Site-directed mutagenesis of lysine 193 in Escherichia coli isocitrate lyase by use of unique restriction enzyme site elimination.

By a newly developed double-stranded mutagenesis technique, histidine (H), glutamate (E), arginine (R) and leucine (L) have been substituted for the lysyl 193 residue (K-193) in isocitrate lyase from Escherichia coli. The substitutions for this residue, which is present in a highly conserved, cationic region, significantly affect both the Km for Ds-isocitrate and the apparent kcat of isocitrate lyase. Specifically, the conservative substitutions, K-193-->H (K193H) and K193R, reduce catalytic activity by ca. 50- and 14-fold, respectively, and the nonconservative changes, K193E and K193L, result in assembled tetrameric protein that is completely inactive. The K193H and K193R mutations also increase the Km of the enzyme by five- and twofold, respectively. These results indicate that the cationic and/or acid-base character of K193 is essential for isocitrate lyase activity. In addition to the noted effects on enzyme activity, the effects of the mutations on growth of JE10, an E. coli strain which does not express isocitrate lyase, were observed. Active isocitrate lyase is necessary for E. coli to grow on acetate as the sole carbon source. It was found that a mutation affecting the activity of isocitrate lyase similarly affects the growth of E. coli JE10 on acetate when the mutated plasmid is expressed in this organism. Specifically, the lag time before growth increases over sevenfold and almost twofold for E. coli JE10 expressing the K193H and K193R isocitrate lyase variants, respectively. In addition, the rate of growth decreases by almost 40-fold for E. coli JE10 cells expressing form K193H and ca. 2-fold for those expressing the K193R variants. Thus, the onset and rate of E. coli growth on acetate appears to depend on isocitrate lyase activity.

A facile method to determine the CO2/O 2 specificity factor for ribulose bisphosphate carboxylase/oxygenase.

A rapid method to determine the CO2/O2 specificity factor of ribulose 1,5-bisphosphate carboxylase/oxygenase is presented. The assay measures the amount of CO2 and O2 fixation at varying CO2/O2 ratios to determine the relative rates of each reaction. CO2 fixation is measured by the incorporation of the moles of(14)CO2 into 3-phosphoglycerate, while O2 fixation is determined by subtraction of the moles of CO2 fixed from the moles of RuBP consumed in each reaction. By analyzing the inorganic phosphate specifically hydrolyzed from RuBP under alkaline conditions, the amount of RuBP present before and after catalysis by rubisco can be determined.

Purification and characterization of chaperonin 10 from Chromatium vinosum.

Chromatium vinosum contains a polypeptide that is functionally and structurally similar to the Escherichia coli chaperonin 10. The protein has been purified to homogeneity by sucrose density gradient centrifugation followed by gel filtration using a Bio-Gel A-1.5 m column. The molecular mass of chaperonin 10, as determined by gel filtration or nondenaturing polyacrylamide gel electrophoresis, is 95 kDa. The oligomer is composed of seven or eight subunits. Comparisons of the overall amino acid composition and N-terminal sequences among chaperonin 10 species from C. vinosum and E. coli reflect a high degree of similarity. A physical association between chaperonins 60 and 10 from C. vinosum, in vitro, is supported by three experimental approaches. First, the proteins form a stable binary complex in sucrose density gradients, gel filtration chromatography, and nondenaturing polyacrylamide gel electrophoresis, solely in the presence of ATP and Mg2+. Second, chaperonin 10 from C. vinosum binds, selectively, to a chaperonin 60-coupled Affi-Gel 10 matrix column. Third, a slight molar excess of chaperonin 10 is able to abolish, almost completely, the ATPase in chaperonin 60. The rate for ATPase activity of chaperonin 60 from C. vinosum is enhanced when supplemented with monovalent cations.

Serine-376 contributes to the binding of substrate by ribulose-bisphosphate carboxylase/oxygenase from Anacystis nidulans.

Site-directed mutagenesis was used to change Ser376 in the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans to Cys, Thr, or Ala. When expressed in Escherichia coli and purified, the mutant enzymes exhibited carboxylase activities that were reduced by 99% or more with respect to the activity of the wild-type enzyme. The Km values for ribulose bisphosphate at pH 8.0, 30 degrees C, were elevated from 46 microM for wild-type enzyme to 287, 978, and 81 microM for mutants in which Cys, Thr, or Ala, respectively, replaced Ser376. The Cys and Thr variants were almost devoid of oxygenase activity whereas the Ala variant had 16% as much oxygenase as wild-type enzyme, suggesting that this mutation had greatly elevated the oxygenase:carboxylase ratio.