Choline Acetyltransferase-Deficient Mutants of the Nematode CAENORHABDITIS ELEGANS

We have identified five independent allelic mutations, defining the gene cha-1, that result in decreased choline acetyltransferase (ChAT) activity in Caenorhabditis elegans. Four of the mutant alleles, when homozygous, lead to ChAT reductions of>98%, as well as recessive phenotypes of uncoordinated behavior, small size, slow growth and resistance to cholinesterase inhibitors. Animals homozygous for the fifth allele retain approximately 10% of the wild-type enzyme level; purified enzyme from this mutant has altered K_m values for both choline and acetyl-CoA and is more thermolabile than the wild-type enzyme. These qualitative alterations, together with gene dosage data, argue that cha-1 is the structural gene for ChAT. cha-1 has been mapped to the left arm of linkage group IV and is within 0.02 map unit of the gene unc-17, mutant alleles of which lead to all of the phenotypes of cha-1 mutants except for the ChAT deficiency. Extensive complementation studies of cha-1 and unc-17 alleles reveal a complex complementation pattern, suggesting that both loci may be part of a single complex gene.     

Guanosine triphosphate and colchicine affect the activity and the polymeric state of acetyl-CoA carboxylase

Acetyl-CoA carboxylase was purified 300-fold from rat liver, in the absence of added citrate, by precipitation from an 18,000g supernatant in the presence of Triton X-100 at 105,000g and 20 °C, followed by chromatography on phosphocellulose. Acetyl-CoA carboxylase activity in this preparation was activated by preincubation with GTP (0.1–2.0 mm) and with citrate (20 mm). Colchicine (10^?6–10^?3m) inhibited enzyme activity and counteracted the effects of GTP and citrate. Sucrose density gradient centrifugation demonstrated that GTP and citrate preincubation promoted the formation of the polymeric, active enzyme, while colchicine engendered disassembly. Preincubation of the purified acetyl-CoA carboxylase at 4 °C caused inactivation and disassembly, which was countered by preincubation at 37 °C in the presence of GTP or citrate. These results suggest that GTP, like citrate, activates acetyl-CoA carboxylase by enhancing the conversion of the protomeric form of the enzyme to its more active, polymeric state.     

Enhancement of the thermostability of a recombinant β-agarase, AgaB, from Zobellia galactanivorans by random mutagenesis

Random mutagenesis was performed on β-agarase, AgaB, from Zobellia galactanivorans to improve its catalytic activity and thermostability. The activities of three mutants E99K, T307I and E99K–T307I were approx. 140, 190 and 200%, respectively, of wild type β-agarase (661 U/mg) at 40°C. All three mutant enzymes were stable up to 50°C and E99K–T307I had the highest thermostability. The melting temperature (T _m) of E99K–T307I, determined by CD spectra, was increased by 5.2°C over that of the wild-type enzyme (54.6°C). Activities of both the wild-type and E99K–T307I enzymes, as well as their overall thermostabilities, increased in 1 mM CaCl₂. The E99K–T307I enzyme was stable at 55°C with 1 mM CaCl₂, reaching 260% of the activity the wild-type enzyme held at 40°C without CaCl₂.     

Cold-active esterase from Psychrobacter sp. Ant300: gene cloning,characterization, and the effects of Gly→Pro substitution near the active site on its catalytic activity and stability

The gene encoding an esterase (PsyEst) of Psychrobacter sp. Ant300, a psychrophilic bacterium isolated from Antarctic soil, was cloned, sequenced, and expressed in Escherichia coli. PsyEst, which is a member of hormone-sensitive lipase (HSL) group of the lipase/esterase family, is a cold-active, themolabile enzyme with high catalytic activity at low temperatures (5–25 °C), low activation energy (e.g., 4.6 kcal/mol for hydrolysis of p-nitrophenyl butyrate), and a t_1/2 value of 16 min for thermal inactivation during incubation at 40 °C and pH 7.9. A three-dimensional structural model of PsyEst predicted that Gly²⁴⁴ was located in the loop near the active site of PsyEst and that substitution of this amino-acid residue by proline should potentially rigidify the active-site environment of the enzyme. Thus, we introduced the Gly²⁴⁴→Pro substitution into the enzyme. Stability studies showed that the t_1/2 value for thermal inactivation of the mutant during incubation at 40 °C and pH 7.9 was 11.6 h, which was significantly greater than that of the wild-type enzyme. The k_cat/K_m value of the mutant was lower for all substrates examined than the value of the wild type. Moreover, this amino-acid substitution caused a shift of the acyl-chain length specificity of the enzyme toward higher preference for short-chain fatty acid esters. All of these observations could be explained in terms of a decrease in active-site flexibility brought about by the mutation and were consistent with the hypothesis that cold activity and thermolability arise from local flexibility around the active site of the enzyme.     

A <Emphasis Type="Italic">Paenibacillus</Emphasis> sp. dextranase mutant pool with improved thermostability and activity

Random mutagenesis was used to create a library of chimeric dextranase (dex1) genes. A plate-screening protocol was developed with improved thermostability as a selection criterion. The mutant library was screened for active dextranase variants by observing clearing zones on dextran-blue agar plates at 50°C after exposure to 68°C for 2 h, a temperature regime at which wild-type activity was abolished. A number of potentially improved variants were identified by this strategy, five of which were further characterised. DNA sequencing revealed ten nucleotide substitutions, ranging from one to four per variant. Thermal inactivation studies showed reduced (2.9-fold) thermostability for one variant and similar thermostability for a second variant, but confirmed improved thermostability for three mutants with 2.3- (28.9 min) to 6.9-fold (86.6 min) increases in half-lives at 62°C compared to that of the wild-type enzyme (12.6 min). Using a 10-min assay, apparent temperature optima of the variants were similar to that of the wild type (T _opt 60°C). However, one of these variants had increased enzyme activity. Therefore, the first-generation dextranase mutant pool obtained in this study has sufficient molecular diversity for further improvements in both thermostability and activity through recombination (gene shuffling).     

Probing the Role of Cysteine Residues in Glucosamine-1-Phosphate Acetyltransferase Activity of the Bifunctional GlmU Protein from Escherichia coli: Site-Directed Mutagenesis and Characterization of the Mutant Enzymes

The glucosamine-1-phosphate acetyltransferase activity but not the uridyltransferase activity of the bifunctional GlmU enzyme from Escherichia coli was lost when GlmU was stored in the absence of β-mercaptoethanol or incubated with thiol-specific reagents. The enzyme was protected from inactivation in the presence of its substrate acetyl coenzyme A (acetyl-CoA), suggesting the presence of an essential cysteine residue in or near the active site of the acetyltransferase domain. To ascertain the role of cysteines in the structure and function of the enzyme, site-directed mutagenesis was performed to change each of the four cysteines to alanine, and plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged proteins. Whereas the kinetic parameters of the bifunctional enzyme appeared unaffected by the C296A and C385A mutations, 1,350- and 8-fold decreases of acetyltransferase activity resulted from the C307A and C324A mutations, respectively. The K_m values for acetyl-CoA and GlcN-1-P of mutant proteins were not modified, suggesting that none of the cysteines was involved in substrate binding. The uridyltransferase activities of wild-type and mutant GlmU proteins were similar. From these studies, the two cysteines Cys307 and Cys324 appeared important for acetyltransferase activity and seemed to be located in or near the active site.     

Studies on temperature-dependent ultraviolet light-sensitive mutants of bacteriophage T4: the structural gene for T4 endonuclease V.

Mutants of bacteriophage T4 which exhibit increased sensitivity to ultraviolet radiation specifically at high temperature were isolated after mutagenesis with hydroxylamine. At 42 °C the mutants are twice as sensitive to ultraviolet light as T4D, whereas at 30 °C they exhibit survival curves almost identical to that of the wild-type strain. Complementation tests revealed that the mutants possess temperature-sensitive mutations in the v gene.Evidence is presented to show that T4 endonuclease V produced by the mutants is more thermolabile than the enzyme of the wild-type. (1) Extracts of cells infected with the mutants were capable of excising pyrimidine dimers from ultraviolet irradiated T4 DNA at 30 °C, but no selective release of dimers was induced at 42 °C. (2) Endonuclease V produced by the mutant was inactivated more rapidly than was the enzyme from T4D-infected cells when the purified enzymes were incubated in a buffer at 42 °C. From these results it is evident that the v gene is the structural gene for T4 endonuclease V, which plays an essential role in the excision-repair of ultraviolet light-damaged DNA.The time of action of the repair endonuclease was determined by using the mutant. Survival of a temperature-sensitive v mutant, exposed to ultraviolet light, increased when infected cells were incubated at 30 °C for at least ten minutes and then transferred to 42 °C. It appears that repair of DNA proceeds during an early stage of phage development.     

Kinetic modeling of thermal inactivation of the Bacillus sp. protease P7

Data from thermal stability of a keratinolytic protease produced by the Amazon isolate Bacillus sp. P7 was fitted to various mathematical models. Kinetic modeling showed that Weibull distribution was the best equation to describe the residual activity of protease P7 after heat treatment. The effects of temperature on equation parameters and on characteristics of the inactivation curves were evaluated. As expected, faster inactivation was observed at higher temperatures. The critical temperature to accelerate protease decomposition was about 70 °C. The reliable life (t _R) of the enzyme, analogous to the D value, ranged from 1,824 to 8 min at 45–65 °C. Within these temperatures, an increase of 8.81 °C was needed to lower enzyme t _R in one-log unit. Protease P7 is a potentially useful biocatalyst for various industrial bioprocesses, and therefore, kinetic modeling of thermal inactivation addresses an important topic aiming enzyme characterization and applications.     

A mutation in the gene for trigger factor suppresses the defect in cell division in the divE42 mutant of Escherichia coli K12

A total of sixteen spontaneously generated, independent suppressor mutants was isolated from a mutant (divE42) of Escherichia coli K12 that is defective in cell division. One of the suppressor mutants, designated TR4, had a novel phenotype: it was able to grow at 42°?C but not at 32°?C. The Kohara genomic library was screened for complementing clones. Clone 148 was able to complement the mutation responsible for the cold-sensitive phenotype, and the gene for trigger factor (tig), which encodes a ribosome-associated peptidyl-prolyl cis/trans isomerase, was identified as the mutated gene by deletion analysis with the insert DNA from clone 148. DNA sequencing revealed that the mutation in the tig gene of the TR4 suppressor mutant was a single nucleotide insertion (+A) at a distance of 834 nucleotides from the initiation codon for this enzyme. When the wild-type tig gene was introduced into the TR4 suppressor mutant, the bacteria were able to grow at 32°?C but not at 42°?C, an indication that the intergenic suppressor mutation was recessive to the wild-type allele. A model is proposed that accounts for the phenotypes of the divE42 mutant and the TR4 suppressor mutant.     

Engineering a high-performance,metagenomic-derived novel xylanase with improved soluble protein yield and thermostability

The novel termite gut metagenomic-derived GH11 xylanase gene xyl7 was expressed in Escherichia coli BL21, and the purified XYL7 enzyme exhibited high specific activity (6340 U/mg) and broad pH active range of 5.5–10.0. Directed evolution was employed to enhance the thermostability of XYL7; two mutants (XYL7-TC and XYL7-TS) showed a 250-fold increase in half-life at 55 °C, with a 10 °C increase in optimal temperature compared to that of wild-type XYL7. A truncated enzyme (XYL7-Tr3) acquired by protein engineering showed similar catalytic properties as the wild-type, with a tenfold increase in soluble protein yield by the mutant. The reducing sugar produced by XYL7-TC was about fourfold greater than that produced by their parents when incubated with xylan at 60 °C for 4 h. The engineered novel xylanase exhibited superior enzymatic performance and showed promise as an excellent candidate for industrial application due to its high specific activity, stability and soluble protein yield.     

Purification and properties of a citrate synthase from Nitrosomonas sp. TK794 and a comparison with the enzymes of another nitrifying bacteria

Citrate(si)-synthase (citrate oxaloacetate-lyasem EC 4.1.3.7) was purified as an electrophoretically homogeneous protein from an ammonia-oxidizing chemoautotrophic bacterium, Nitrosomonas sp. TK794. The molecular mass of the native enzyme was estimated to be about 287 kDa by gel filtration, whereas SDS-PAGE produced one band with M_r values of 44.7 kDa, suggesting that the enzyme is a hexamer consisting of identical subunits. The isoelectric point of the enzyme was 5.0. The pH and temperature optima for citrate synthase (CS) activity was about 7.5–8.0 and 40°C, respectively. The citrate synthase was stable over a pH range of 6.0–8.5 and up to 40°C. The apparent K_m values for oxaloacetate and acetyl-CoA were about 11 μM and 247 μM, respectively. The activity of the citrate synthase was not inhibited by ATP, NADH or 2-oxoglutarate at 5mM, and was activated by potassium chloride at 0.1–100 mM. The N-terminal amino acid sequence of the enzyme protein was PPQDVATLSPGENKKTIELPILG.     

Impact of cofactor-binding loop mutations on thermotolerance and activity of E. coli transketolase

Improvement of thermostability in engineered enzymes can allow biocatalysis on substrates with poor aqueous solubility. Denaturation of the cofactor-binding loops of Escherichia coli transketolase (TK) was previously linked to the loss of enzyme activity under conditions of high pH or urea. Incubation at temperatures just below the thermal melting transition, above which the protein aggregates, was also found to anneal the enzyme to give an increased specific activity. The potential role of cofactor-binding loop instability in this process remained unclear. In this work, the two cofactor-binding loops (residues 185–192 and 382–392) were progressively mutated towards the equivalent sequence from the thermostable Thermus thermophilus TK and variants assessed for their impact on both thermostability and activity. Cofactor-binding loop 2 variants had detrimental effects on specific activity at elevated temperatures, whereas the H192P mutation in cofactor-binding loop 1 resulted in a two-fold improved stability to inactivation at elevated temperatures, and increased the critical onset temperature for aggregation. The specific activity of H192P was 3-fold and 19-fold higher than that for wild-type at 60 °C and 65 °C respectively, and also remained 2.7-4 fold higher after re-cooling from pre-incubations at either 55 °C or 60 °C for 1 h. Interestingly, H192P was also 2-times more active than wild-type TK at 25 °C. Optimal activity was achieved at 60 °C for H192P compared to 55 °C for wild type. These results show that cofactor-binding loop 1, plays a pivotal role in partial denaturation and aggregation at elevated temperatures. Furthermore, a single rigidifying mutation within this loop can significantly improve the enzyme specific activity, as well as the stability to thermal denaturation and aggregation, to give an increased temperature optimum for activity.     

Effects of temperature changes on thymidine kinase in heat- and cold-sensitive cell-cycle mutants and ‘wild-type’ murine P-815 cells

Two heat-sensitive (arrested in G1 at 39.5°C) and two cold-sensitive (arrested in G1 at 33°C) clonal cell-cycle mutants that had been isolated from the same clone (K 21), of the murine mastocytoma P-815 cell line, were tested for thymidine kinase (EC 2.7.1.21) activity. After shift of mutant cells to the nonpermissive temperature, thymidine kinase activity decreased, and minimal levels (i.e., less than 3% of those observed for ‘wild-type’ K 21 cells at the respective temperature) were attained within 16 h in heat-sensitive and after 3–4 days in cold-sensitive mutants, which is in good agreement with kinetics of accumulation of heat-sensitive and cold-sensitive cells in G1 phase. After return of arrested mutant cells to the permissive temperature, thymidine kinase of heat-sensitive cells increased rapidly and in parallel with entry of cells into the S phase. In cultures of cold-sensitive cells, however, initiation of DNA synthesis preceded the increase of thymidine kinase activity by approx. one cell-cycle time. Thymidine kinase activities in revertants of the heat-sensitive and cold-sensitive mutants were similar to those of ‘wild-type’ cells. In ‘wild-type’ K 21 cells incubated at 39.5°C, thymidine kinase activity was approx. 30% of that at 33°C. This difference is attributable, at least in part, to a higher rate of inactivation of the enzyme at 39.5°C, as determined in cultures incubated with cycloheximide. The rapid increase of thymidine kinase activity that occurred after shift of K 21 cells and of arrested heat-sensitive mutant cells from 39.5°C to 33°C was inhibited by actinomycin D and cycloheximide.     

Stimulation of choline acetyltransferase activity by ethanol in in vitro preparations of rat cerebrum

Choline acetyltransferase activity in homogenates, or in partially purified extracts of rat brain cerebra, was increased by 11–37% in the presence of ethanol when incubated at 38°C with [¹⁴C] acetyl-CoA, choline chloride and alcohol concentrations of 0.17M to 1.02M. In preincubation experiments with enzyme preparations and ethanol, inactivation of the enzyme by the alcohol, which occurs at incubation times longer than 20 minutes, could be at least partially prevented by the addition of certain components of the incubation mixture to the preincubation mixture.     

Temperature-sensitive mutants blocked in the folding or subunit assembly of the bacteriophage P22 tail spike protein: II. Active mutant proteins matured at 30 °C

Little is known of the molecular mechanisms by which temperature-sensitive mutations interfere with the formation of biologically active proteins. We have studied the effects of such mutations at 13 different sites on the properties of the multifunctional tail spike protein of bacteriophage P22, a thermostable structural protein composed of 76,000 M_r chains.Using multiple mutant strains blocked in capsid assembly, we have examined the free mutant tail spikes that accumulate in active form at permissive temperature. When assayed for the ability to bind to phage heads at the restrictive temperature, the mutant proteins were as active as the wild type. Similarly, when assayed for the ability to adsorb to bacteria at restrictive temperature, the mutant proteins were as active as the wild type. Thus the temperature-sensitive phenotypes of the mutants are not due to the thermolability of these functions in the mature mutant protein.The wild-type protein is heat-resistant, requiring incubation at 90 °C, to give a half-time of inactivation of ten minutes. The 13 ts mutant proteins, once matured at 30 °C, were as resistant as the wild-type protein to inactivation at elevated temperatures.Though the mature wild-type protein is heat stable, its maturation is heat-sensitive; the number of polypeptide chains synthesized at 30 °C and 39 °C is the same, but the yield of active tail spikes at 39 °C is only 25% of the yield at 30 °C.The results show that the amino acid substitutions in the mutant proteins, though lethal for the formation of the virus at 39 °C, do not affect the thermostability of the mature tail spike protein formed at 30 °C. They may act by destabilizing thermolabile intermediates in the folding or subunit assembly of the tail spike protein.     

Purification and characterization of a mutant ATP phosphoribosyltransferase hypersensitive to histidine feedback inhibition.

A mutant form of ATP phosphoribosyltranferase (EC 2.4.2.17), hisG1708c, which results in abnormally slow growth of Salmonella typhimurium at 20 °C was purified to homogeneity and kinetic and chemical behavior were characterized. Initial velocity steady-state substrate kinetics of wild-type and mutant enzymes at 37 °C were consistent with sequential kinetics and demonstrated that standard assay concentrations of substrates were sufficient to substantially saturate both enzymes. Nearly time-independent inhibition by histidine at 37 °C could be obtained only after incubation in the presence of product and histidine. Studies at 37 °C showed that the mutant enzyme is 24 times more sensitive to histidine than the wild type in a negatively cooperative manner instead of the positively cooperative manner seen for wild type. Pure mutant enzyme exhibits two major electrophoretic species of native enzyme. Although one less cysteine is titratable in native mutant enzyme, the amino acid compositions of mutant and wild-type enzymes are similar. Histidine produces an ultraviolet difference spectrum in mutant enzyme closely resembling that produced in wild type. Binding of histidyl-tRNA to mutant enzyme is substantially inhibited by histidine. It is concluded that the hisG1708c mutation alters some conformational processes coupled to the histidine binding site while not affecting others.     

Site-directed mutagenesis of an Aspergillus niger xylanase B and its expression,purification and enzymatic characterization in Pichia pastoris

Xylanase is an important industrial enzyme. In this research, to improve the thermostability and biochemical properties of a xylanase from Aspergillus niger F19, five arginine substitutions and a disulfide bond were introduced by site-directed mutagenesis. The wild-type gene xylB and the mutant gene xylCX8 were expressed in Pichia pastoris. Compare to those of the wild-type enzyme, the optimal reaction temperature for the mutant enzyme increased from 45 °C to 50 °C, the half-life of the mutant enzyme extended from 10 min to 180 min, and the specific activity increased from 2127 U/mg to 3330 U/mg. However, the V_max and K_m of the mutant xylanase decreased. The enzyme activity in broth obtained from shake flask cultures could be induced to 1850 U/mL in 7 days, which is higher than results reported previously. Furthermore, the highest achievable enzyme activity was 7340 U/mL from 140 g/L of biomass in a 3 L fermentor used in our study.     

Synthesis of tryptophanase in Escherichia coli: Isolation and characterization of a structural-gene mutant and two regulatory mutants

Summary A mutant of E. coli has been isolated that is temperature-sensitive in respect of tryptophanase. When incubated at 60°C, cell-free extracts of the mutant suffer inactivation of enzyme activity much more rapidly than similar extracts of the wild type. After lysogeny with a specialized transducing phage carrying the wild-type tryptophanase gene, the mutant is able to synthesize tryptophanase that is wild-type in its response to treatment at 60°C. It is concluded that the mutation lies in the structural gene for the enzyme.Two further mutants have been isolated that synthesize tryptophanase constitutively. One mutation renders synthesis of the enzyme indifferent to the presence of inducer; the other mutation allows synthesis of the enzyme in the absence of inducer at about 35% of the fully induced wild-type rate. Neither mutation alleviates catabolite repression. Genetic mapping shows that the constitutive mutations lie very close to the structural-gene mutation, on the side of the structural gene distant from bglR.     

Purification and characterization of human lung calmodulin-independent cyclic AMP phosphodiesterase

A high-affinity calmodulin-independent cyclic AMP phosphodiesterase was purified to homogeneity from human lung tissue. This enzyme has a molecular weight of 60,000, a sedimentation coefficient of 3.2–3.4 S, and an isoelectric pH of 4.6–4.8. Neither Ca²⁺ nor calmodulin (in the presence or absence of added Ca²⁺) stimulates the enzymatic activity. This enzyme appears to be very similar to that described previously from dog kidney (W. J. Thompson, P. M. Epstein, and S. J. Strada, (1979) Biochemistry18, 5228–5237). Hydrolysis of cyclic AMP is greatly enhanced by Mg²⁺ (25–30× at 10 mm Mg²⁺) and Mn²⁺ (20× at 10 mm Mn²⁺). Zn²⁺, Cu²⁺, and Co²⁺ are ineffective at these concentrations. Cyclic AMP is the exclusive substrate with a K_m of 0.7–0.8 μm. The I₅₀ of cyclic GMP is 1 mm using 1 μm cyclic AMP as substrate. In contrast, aminophylline, MIX, and SQ 20009 have I₅₀s of 0.28, 0.021, and 0.001 mm, respectively). The purified enzyme is susceptible to temperature inactivation and protease degradation. Significant (10%) inhibition is seen at 37 °C for 20 min. Trypsin, at 0.1 μg/ml, destroys 50% of the activity in 30 min at 25 °C. Our observations concerning its lability to temperature and proteases coupled with its lack of response to calmodulin suggest this enzyme is a basic catalytic subunit of other cyclic AMP phosphodiesterases present within human lung tissue.