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.     

Site-directed mutagenesis of active site residues in Bacillus subtilis alpha-amylase.

Site-directed mutagenesis of Bacillus subtilis N7 alpha-amylase has been performed to evaluate the roles of the active site residues in catalysis and to prepare an inactive catalytic-site mutant that can form a stable complex with natural substrates. Mutation of Asp-176, Glu-208, and Asp-269 to their amide forms resulted in over a 15,000-fold reduction of its specific activity, but all the mutants retained considerable substrate-binding abilities as estimated by gel electrophoresis in the presence of soluble starch. Conversion of His-180 to Asn resulted in a 20-fold reduction of kcat with a 5-fold increase in Km for a maltopentaose derivative. The relative affinities for acarbose vs. maltopentaose were also compared between the mutants and wild-type enzyme. The results are consistent with the roles previously proposed in Taka-amylase A and porcine pancreatic alpha-amylase based on their X-ray crystallographic analyses, although different pairs had been assigned as catalytic residues for each enzyme. Analysis of the residual activity of the catalytic-site mutants by gel electrophoresis has suggested that it derived from the wild-type enzyme contaminating the mutant preparations, which could be removed by use of an acarbose affinity column; thus, these mutants are completely devoid of activity. The affinity-purified mutant proteins should be useful for elucidating the complete picture of the interaction of this enzyme with starch.     

Site-directed mutagenesis of active site residues of phosphite dehydrogenase

Phosphite dehydrogenase (PTDH) catalyzes the unusual oxidation of phosphite to phosphate with the concomitant reduction of NAD(+) to NADH. PTDH shares significant amino acid sequence similarity with D-hydroxy acid dehydrogenases (DHs), including strongly conserved catalytic residues His292, Glu266, and Arg237. Site-directed mutagenesis studies corroborate the essential role of His292 as all mutants of this residue were completely inactive. Histidine-selective inactivation studies with diethyl pyrocarbonate provide further evidence regarding the importance of His292. This residue is most likely the active site base that deprotonates the water nucleophile. Kinetic analysis of mutants in which Arg237 was changed to Leu, Lys, His, and Gln revealed that Arg237 is involved in substrate binding. These results agree with the typical role of this residue in D-hydroxy acid DHs. However, Glu266 does not play the typical role of increasing the pK(a) of His292 to enhance substrate binding and catalysis as the Glu266Gln mutant displayed an increased k(cat) and unchanged pH-rate profile compared to those of wild-type PTDH. The role of Glu266 is likely the positioning of His292 and Arg237 with which it forms hydrogen bonds in a homology model. Homology modeling suggests that Lys76 may also be involved in substrate binding, and this postulate is supported by mutagenesis studies. All mutants of Lys76 display reduced activity with large effects on the K(m) for phosphite, and Lys76Cys could be chemically rescued by alkylation with 2-bromoethylamine. Whereas a positively charged residue is absolutely essential for activity at the position of Arg237, Lys76 mutants that lacked a positively charged side chain still had activity, indicating that it is less important for binding and catalysis. These results highlight the versatility of nature's catalytic scaffolds, as a common framework with modest changes allows PTDH to catalyze its unusual nucleophilic displacement reaction and d-hydroxy acid DHs to oxidize alcohols to ketones.     

Site-directed mutagenesis of the calcium-binding site of blood coagulation factor XIIIa.

Blood coagulation factor XIIIa is a calcium-dependent enzyme that covalently ligates fibrin molecules during blood coagulation. X-ray crystallography studies identified a major calcium-binding site involving Asp(438), Ala(457), Glu(485), and Glu(490). We mutated two glutamic acid residues (Glu(485) and Glu(490)) and three aspartic acid residues (Asp(472), Asp(476), and Asp(479)) that are in close proximity. Alanine substitution mutants of these residues were constructed, expressed, and purified from Escherichia coli. The K(act) values for calcium ions increased by 3-, 8-, and 21-fold for E485A, E490A, and E485A,E490A, respectively. In addition, susceptibility to proteolysis was increased by 4-, 9-, and 10-fold for E485A, E490A, and E485A,E490A, respectively. Aspartic acids 472, 476, and 479 are not involved directly in calcium binding since the K(act) values were not changed by mutagenesis. However, Asp(476) and Asp(479) are involved in regulating the conformation for exposure of the secondary thrombin cleavage site. This study provides biochemical evidence that Glu(485) and Glu(490) are Ca(2+)-binding ligands that regulate catalysis. The binding of calcium ion to this site protects the molecule from proteolysis. Furthermore, Asp(476) and Asp(479) play a role in modulating calcium-dependent conformational changes that cause factor XIIIa to switch from a protease-sensitive to a protease-resistant molecule.     

Site-directed mutagenesis of the active site cysteine in Klebsiella aerogenes urease.

Cysteine 319 in the large subunit of Klebsiella aerogenes urease was identified as an essential catalytic residue based on chemical modification studies (Todd, M.J., and Hausinger, R.P. (1991) J. Biol. Chem. 266, 24327-24331). Through site-directed mutagenesis, this cysteine has been changed independently to alanine, serine, aspartate, and tyrosine. None of these mutations (C319A, C319S, C319D, and C319Y, respectively) affected the size or level of synthesis of the urease subunits as monitored by polyacrylamide gel electrophoresis. The wild type enzyme and each of the mutant proteins was purified and their properties were compared. The C319Y protein possessed no detectable activity, while activity was reduced in C319A, C319S, and C319D to 48, 4.5, and 0.03% of wild type levels under normal assay conditions. All of the active mutants had a small increase in Km when compared to the wild type value. The active mutants displayed a greatly reduced sensitivity to inactivation by iodoacetamide in comparison to the wild type enzyme, confirming our previous assignment of the essential cysteine to this residue based on active site peptide mapping. In contrast to the wild type enzyme, inactivation of the mutant proteins was not affected by the presence of the competitive inhibitor phosphate, suggesting that the remaining slow rate of iodoacetamide inactivation is due to modification away from the active site. The pH dependence of urease activity was substantially altered in the active mutants with C319S and C319D showing a pH optimum near 5.2, and C319A near 6.7, compared to the pH 7.75 optimum of wild type urease. These data are consistent with Cys-319 facilitating catalysis at neutral and basic pH values by participating as a general acid.     

Site-directed mutagenesis in the effector site of Escherichia coli phosphofructokinase

A new vector for the expression of phosphofructokinase (pfk-1) was constructed with pEMBL, which allows reliable, inducible, high-expression, and facile mutagenesis of the gene. Two mutants in the effector site of the enzyme were produced by site-specific mutagenesis of residue Tyr-55 to assess the role of its side chain in binding an allosteric inhibitor, phosphoenolpyruvate (PEP), and an activator, guanosine 5'-diphosphate (GDP): Tyr-55----Phe-55 and Try-55----Gly-55. The dissociation constant of PEP from the T state is unaffected by the mutations. Mutation of Tyr-55----Phe-55 only slightly increases the dissociation constant of GDP from the R state, indicating a minimal involvement of the hydroxyl group in binding. A 5.5-fold increase in the dissociation constant of GDP on the mutation of Tyr-55----Gly-55 suggests a small hydrophobic interaction of the aromatic ring of the tyrosine residue with guanine of GDP.     

A high efficiency method for site-directed mutagenesis with any plasmid

A procedure is described which allows for the site-directed mutagenesis of DNA segments in any double-stranded plasmid with high efficiency. There are no limitations as to the position of the mutation. The protocol involves only simple enzymatic manipulations and no difficult to control operations, such as partial digestions, are required. The method was developed and used to mutagenize two different genes (encoding human interferon-beta and interleukin-2) cloned in a eukaryotic expression vector. For ten mutageneses with different oligodeoxyribonucleotides the average yield of mutants was 60%.     

Site-directed mutagenesis of Escherichia coli succinyl-CoA synthetase. beta-Cys325 is a nonessential active site residue

Chemical modification experiments have shown that sulfhydryl groups play an important role in the mechanism of action of Escherichia coli succinyl-CoA synthetase. One of these sulfhydryl groups has been localized in the beta-subunit of the enzyme using the coenzyme A affinity analog, CoA disulfide-S,S-dioxide (Collier, G. E., and Nishimura, J. S. (1978) J. Biol. Chem. 253, 4938-4943). Recently, it has been shown that the reactive sulfhydryl group resides in Cys325 (Nishimura, J. S., Mitchell, T., Ybarra, J., and Matula, J. M., submitted to Eur. J. Biochem. for publication). In the present study, we have changed Cys325 to a glycine residue using the technique of site-directed mutagenesis and have purified the mutant enzyme to homogeneity. The resulting mutant enzyme is 83% as active as wild type enzyme. In contrast to wild type succinyl-CoA synthetase, the mutant is refractory to chemical modification by CoA disulfide-S,S-dioxide and methyl methanethiolsulfonate. It is also less reactive with N-ethylmaleimide. Thus, beta-Cys325 is a nonessential active site residue.     

The unique ligand-binding pocket for the human prostacyclin receptor. Site-directed mutagenesis and molecular modeling

The human prostacyclin receptor is a seven-transmembrane alpha-helical G-protein coupled receptor, which plays important roles in both vascular smooth muscle relaxation as well as prevention of blood coagulation. The position of the native ligand-binding pocket for prostacyclin as well as other derivatives of the 20-carbon eicosanoid, arachidonic acid, has yet to be determined. Through the use of prostanoid receptor sequence alignments, site-directed mutagenesis, and the 2.8-A x-ray crystallographic structure of bovine rhodopsin, we have developed a three-dimensional model of the agonist-binding pocket within the seven-transmembrane (TM) domains of the human prostacyclin receptor. Upon mutation to alanine, 11 of 29 candidate residues within TM domains II, III, IV, V, and VII exhibited a marked decrease in agonist binding. Of this group, four amino acids, Arg-279 (TMVII), Phe-278 (TMVII), Tyr-75 (TMII), and Phe-95 (TMIII), were identified (via receptor amino acid sequence alignment, ligand structural comparison, and computer-assisted homology modeling) as having direct molecular interactions with ligand side-chain constituents. This binding pocket is distinct from that of the biogenic amine receptors and rhodopsin where the native ligands (also composed of a carbon ring and a carbon chain) are accommodated in an opposing direction. These findings should assist in the development of novel and highly specific ligands including selective antagonists for further molecular pharmacogenetic studies of the human prostacyclin receptor.     

Site-directed mutagenesis identifies catalytic residues in the active site of Escherichia coli phosphofructokinase.

Six active site mutants of Escherichia coli phosphofructokinase have been constructed and characterized using steady-state kinetics. All but one of the mutants (ES222) have significantly lower maximal activity, implicating these residues in the catalytic process. Replacement of Asp127, the key catalytic residue in the forward reaction with Glu, results in an enzyme with wild-type cooperative and allosteric behavior but severely decreased Fru6P binding. Replacement of the same residue with Tyr abolishes cooperativity while retaining sensitivity to allosteric inhibition and activation. Thus, this mutant has uncoupled homotropic from heterotropic allostery. Mutation of Asp103 to Ala results in an enzyme which retains wild-type Fru6P-binding characteristics with reduced activity. GDP, which allosterically activates the wild-type enzyme, acts as a mixed inhibitor for this mutant. Mutation of Thr125 to Ala and Asp129 to Ser produces mutants with impaired Fru6P binding and decreased cooperativity. In the presence of the activator GDP, both these mutants display apparent negative cooperativity. In addition, ATP binding is now allosterically altered by GDP. These results extend the number of active site residues known to participate in the catalytic process and help to define the mechanisms behind catalysis and homotropic and heterotropic allostery.     

Site-directed mutagenesis of firefly luciferase active site amino acids: a proposed model for bioluminescence color.

Under physiological conditions firefly luciferase catalyzes the highly efficient emission of yellow-green light from the substrates luciferin, Mg-ATP, and oxygen. In nature, bioluminescence emission by beetle luciferases is observed in colors ranging from green (approximately 530 nm) to red (approximately 635 nm), yet all known luciferases use the same luciferin substrate. In an earlier report [Branchini, B. R., Magyar, R. M., Murtiashaw, M. H., Anderson, S. M., and Zimmer, M. (1998) Biochemistry 37, 15311-15319], we described the effects of mutations at His245 on luciferase activity. In the context of molecular modeling results, we proposed that His245 is located at the luciferase active site. We noted too that the H245 mutants displayed red-shifted bioluminescent emission spectra. We report here the construction and purification of additional His245 mutants, as well as mutants at residues Lys529 and Thr343, all of which are stringently conserved in the beetle luciferase sequences. Analysis of specific activity and steady-state kinetic constants suggested that these residues are involved in luciferase catalysis and the productive binding of substrates. Bioluminescence emission spectroscopy studies indicated that point mutations at His245 and Thr343 produced luciferases that emitted light over the color range from green to red. The results of mutational and biochemical studies with luciferase reported here have enabled us to propose speculative mechanisms for color determination in firefly bioluminescence. An essential role for Thr343, the participation of His245 and Arg218, and the involvement of bound AMP are indicated.     

运用新型的反向PCR策略高效构建基因的点突变体

基因的点突变体在其结构和功能研究中发挥非常关键的作用,如何高效、经济地构建基因点突变体是许多分子生物学研究遇到的棘手问题.以PIAS3点突变体的构建为对象,设计了新型的以反向PCR为基础的点突变构建流程,获得的点突变体质粒经测序后均与预期相符,并在293T细胞内得到了正确表达.以上结果表明,该实验设计方案能够高效、方便地用于基因点突变体的构建,为进一步研究它们的分子功能打下了基础.     

Site-directed mutagenesis by combination of homologous recombination and DpnI digestion of the plasmid template in Escherichia coli

A rapid site-directed mutagenesis strategy using homologous recombination and DpnI digestion of the template in Escherichia coli is described. Briefly, inverse polymerase chain reaction amplification of the entire circular plasmid was performed by mutagenic primers with overlapping sequences ( approximately 15 bp) for generating PCR products with approximately 15 bp of homology on the terminal ends. On direct transformation of the amplified PCR products into restriction endonuclease DpnI-expressing E. coli BUNDpnI, homologous recombination occurs in E. coli while the original templates are removed via DpnI digestion in vivo, thus yielding clones harboring mutated circular plasmids. Nearly 100% efficiency was attained when this strategy was used to modify DNA sequences.     

Site-directed mutagenesis of the spinach acyl carrier protein-I prosthetic group attachment site

Site-directed mutagenesis was used to change the phosphopantetheine attachment site (Ser38) of spinach acyl carrier protein I (ACP-I) from a serine to a threonine or cysteine residue. 1. Although the native ACP-I is fully phosphopantethenylated when expressed in Escherichia coli, the TH-ACP-I and CY-ACP-I mutants were found to be completely devoid of the phosphopantetheine group. Therefore, the E. coli holoACP synthase requires serine for in vivo phosphopantetheine addition to spinach ACP-I. 2. Spinach holoACP synthase was completely inactive in vitro with either the TH-ACP-I or CY-ACP-I mutants. In addition, TH-ACP-I and CY-ACP-I were strong inhibitors of spinach holoACP synthase. 3. The mutant ACPs were weak or ineffective as inhibitors of spinach fatty acid synthesis and spinach oleoyl-ACP hydrolase. 4. Compared to holoACP-I, the mutant apoACP-I analogs had: (a) altered mobility in SDS and native gel electrophoresis, (b) altered binding to anti-(spinach ACP-I) antibodies and (c) altered isoelectric points. The combined physical, immunological and enzyme inhibition data indicate that attachment of the phosphopantheine prosthetic group alters ACP conformation.     

Site-directed mutagenesis at the active site Trp120 of Aspergillus awamori glucoamylase

Trp120 of Aspergillus awamori glucoamylase has previously been shown by chemical modification to be essential for activity and tentatively to be located near subsite 4 of the active site. To further test its role, restriction sites were inserted in the cloned A.awamori gene around the Trp120 coding region, and cassette mutagenesis was used to replace it with His, Leu, Phe and Tyr. All four mutants displayed 2% or less of the maximal activity (kcat) of wild-type glucoamylase towards maltose and maltoheptaose. Michaelis constants (KM) of mutants decreased 2- to 3-fold for maltose and were essentially unchanged for maltoheptaose compared with the wild type, except for a greater than 3-fold decrease for maltoheptaose with the Trp120----Tyr mutant. This mutant also bound isomaltose more strongly and had more selectivity for its hydrolysis than wild-type glucoamylase. A subsite map generated from malto-oligosaccharide substrates having 2-7 D-glucosyl residues indicated that subsites 1 and 2 had greater affinity for D-glucosyl residues in the Trp120----Tyr mutant than in wild-type glucoamylase. These results suggest that Trp120 from a distant subsite is crucial for the stabilization of the transition-state complex in subsites 1 and 2.     

Site-directed mutagenesis of a conserved region of the 5-enolpyruvylshikimate-3-phosphate synthase active site

The active site of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) has been probed using site-directed mutagenesis and inhibitor binding techniques. Replacement of a specific glycyl with an alanyl or a prolyl with a seryl residue in a highly conserved region confers glyphosate tolerance to several bacterial and plant EPSPS enzymes, suggesting a high degree of structural conservation between these enzymes. The glycine to alanine substitution corresponding to Escherichia coli EPSPS G96A increases the Ki(app) (glyphosate) of petunia EPSPS 5000-fold while increasing the Km(app)(phosphoenolpyruvate) about 40-fold. Substitution of this glycine with serine, however, abolishes EPSPS activity but results in the elicitation of a novel EPSP hydrolase activity whereby EPSP is converted to shikimate 3-phosphate and pyruvate. This highly conserved region is critical for the interaction of the phosphate moiety of phosphoenolpyruvate with EPSPS.     

Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase

The active site of porphobilinogen (PBG)1 deaminase (EC 4.3.1.8) from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-224, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA- strain of E. coli the enzyme was enriched from [5-13C]ALA and examined by 1H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked head to tail and terminating in a CH2-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I. The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-[2,11-13C2]PBG reveals that the aninomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the alpha-free pyrrole. NMR spectroscopy of the ES2 complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the alpha-free pyrrole position of the enzyme. A mechanistic rationale for deaminase is presented.