Single amino-acid substitution in the N-terminal arm altered the tetramer stability of rat muscle lactate dehydrogenase A

Lactate dehydrogenase A (LDHA) is a well-characterized tetrameric enzyme. Its N-terminal arm, comprised of an α-helix and a β-strand, was suggested to be essential for subunit interactions. To examine the critical amino acid residues in the N-terminus involved in the subunit association, two single-point mutants, Leu3Pro (L3P) and Ile8Glu (I8E), have been constructed. We compared the stability of WT-LDHA (WT) and its variants by unfolding experiments. For WT, a dimeric but inactive intermediate was observed by size-exclusion chromatography at 0.6–0.8 mol/L GdmCl. Leu3Pro exists in an active tetrameric structure in aqueous solution as WT does, but it dissociates into dimers under lower concentration of GdmCl (0.2 mol/L). In aqueous solution, the Ile8Glu variant exists predominantly in the dimeric form with increased K_M and decreasedk _cat as compared with those of WT and L3P. However, the activity of Ile8Glu increases significantly in the presence of sodium sulfate. In conclusion, two mutants are less stable than WT in oligomer structure. Results also support the fact that some residues in the N-terminal arm, especially the Leu8 in the β-structure, contribute the important binding energies to the dimerization of dimers, which might affect the assembly of the enzyme as well as the catalytic function.     

Role of glutamate-52 in the mechanism of L-lactate dehydrogenase from Bacillus stearothermophilus

A single residue of the NAD(H)-dependent lactate dehydrogenase (LDH) from Bacillus stearothermophilus has been changed in order to decrease substrate inhibition. The conserved aspartic acid residue at position 52 was replaced by glutamate using site-directed mutagenesis. The effect on substrate inhibition was measured. In the glutamate-52 mutant substrate inhibition is decreased twofold.     

Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface

The stability of tetrameric malate dehydrogenase from the green phototrophic bacterium Chloroflexus aurantiacus (CaMDH) is at least in part determined by electrostatic interactions at the dimer-dimer interface. Since previous studies had indicated that the thermal stability of CaMDH becomes lower with increasing pH, attempts were made to increase the stability by removal of (excess) negative charge at the dimer-dimer interface. Mutation of Glu165 to Gln or Lys yielded a dramatic increase in thermal stability at pH 7.5 (+23.6 -- + 23.9 degrees C increase in apparent t(m)) and a more moderate increase at pH 4.4 (+4.6 -- + 5.4 degrees C). The drastically increased stability at neutral pH was achieved without forfeiture of catalytic performance at low temperatures. The crystal structures of the two mutants showed only minor structural changes close to the mutated residues, and indicated that the observed stability effects are solely due to subtle changes in the complex network of electrostatic interactions in the dimer-dimer interface. Both mutations reduced the concentration dependency of thermal stability, suggesting that the oligomeric structure had been reinforced. Interestingly, the two mutations had similar effects on stability, despite the charge difference between the introduced side-chains. Together with the loss of concentration dependency, this may indicate that both E165Q and E165K stabilize CaMDH to such an extent that disruption of the inter-dimer electrostatic network around residue 165 no longer limits kinetic thermal stability.     

Charge balance in the alpha-hydroxyacid dehydrogenase vacuole: an acid test.

The proposal that the active site vacuole of NAD(+)-S-lactate dehydrogenase is unable to accommodate any imbalance in electrostatic charge was tested by genetically manipulating the cDNA coding for human muscle lactate dehydrogenase to make a protein with an aspartic acid introduced at position 140 instead of the wild-type asparagine. The Asn 140-Asp mutant enzyme has the same kcat as the wild type (Asn 140) at low pH (4.5), and at higher pH the Km for pyruvate increases 10-fold for each unit increase in pH up to pH 9. We conclude that the anion of Asp 140 is completely inactive and that it binds pyruvate with a Km that is over 1,000 times that of the Km of the neutral, protonated aspartic-140. Experimental results and molecular modeling studies indicate the pKa of the active site histidine-195 in the enzyme-NADH complex is raised to greater than 10 by the presence of the anion at position 140. Energy minimization and molecular dynamics studies over 36 ps suggest that the anion at position 140 promotes the opening of and the entry of mobile solvent beneath the polypeptide loop (98-110), which normally seals off the internal active site vacuole from external bulk solvent.     

Protein stability and electrostatic interactions between solvent exposed charged side chains

To investigate the contribution to protein stability of electrostatic interactions between charged surface residues, we have studied the effect of substituting three negatively charged solvent exposed residues with their side-chain amide analogs in bovine calbindin D9k--a small (Mr 8,500) globular protein of the calmodulin superfamily. The free energy of urea-induced unfolding for the wild-type and seven mutant proteins has been measured. The mutant proteins have increased stability towards unfolding relative to the wild-type. The experimental results correlate reasonably well with theoretically calculated relative free energies of unfolding and show that electrostatic interactions between charges on the surface of a protein can have significant effects on protein stability.     

Variation in Bitterness Potency When Introducing Gly-Gly Residue into Bitter Peptides

We previously reported that Gly-Gly-Arg-Pro and Arg-Pro-Gly-Gly, the derivatives of a bitter peptide Arg-Pro, had no bitterness although Gly-Arg-Pro and Arg-Pro-Gly had a bitter taste at the same level as that of Arg-Pro. To elucidate the mechanism of elimination of bitterness by the introduction of the Gly-Gly residues, we synthesized Gly-Gly derivatives of other bitter peptides such as Phe-Phe, Val-Val-Val, and Arg-Pro-Phe-Phe, and examined the effectiveness of Gly-Gly residues in eliminating bitterness. We suggest that, for Arg-Pro and Val-Val-Val, the Gly-Gly residue might prevent a hydrophobic group from binding to a taste receptor.     

Differences in the roles of conserved glutamic acid residues in the active site of human class 3 and class 2 aldehyde dehydrogenases.

Although the three-dimensional structure of the dimeric class 3 rat aldehyde dehydrogenase has recently been published (Liu ZJ et al., 1997, Nature Struct Biol 4:317-326), few mechanistic studies have been conducted on this isoenzyme. We have characterized the enzymatic properties of recombinant class 3 human stomach aldehyde dehydrogenase, which is very similar in amino acid sequence to the class 3 rat aldehyde dehydrogenase. We have determined that the rate-limiting step for the human class 3 isozyme is hydride transfer rather than deacylation as observed for the human liver class 2 mitochondrial enzyme. No enhancement of NADH fluorescence was observed upon binding to the class 3 enzyme, while fluorescence enhancement of NADH has been previously observed upon binding to the class 2 isoenzyme. It was also observed that binding of the NAD cofactor inhibited the esterase activity of the class 3 enzyme while activating the esterase activity of the class 2 enzyme. Site-directed mutagenesis of two conserved glutamic acid residues (209 and 333) to glutamine residues indicated that, unlike in the class 2 enzyme, Glu333 served as the general base in the catalytic reaction and E209Q had only marginal effects on enzyme activity, thus confirming the proposed mechanism (Hempel J et al., 1999, Adv Exp Med Biol 436:53-59). Together, these data suggest that even though the subunit structures and active site residues of the isozymes are similar, the enzymes have very distinct properties besides their oligomeric state (dimer vs. tetramer) and substrate specificity.     

Structural stability of oligomeric chaperonin 10: the role of two beta-strands at the N and C termini in structural stabilization

Chaperonin 10 (cpn10) is a well-conserved subgroup of the molecular chaperone family. GroES, the cpn10 from Escherichia coli, is composed of seven 10kDa subunits, which form a dome-like oligomeric ring structure. From our previous studies, it was found that GroES unfolded completely through a three-state unfolding mechanism involving a partly folded monomer and that this reaction was reversible. In order to study whether these unfolding-refolding characteristics were conserved in other cpn10 proteins, we have examined the structural stabilities of cpn10s from rat mitochondria (RatES) and from hyperthermophilic eubacteria Thermotoga maritima (TmaES), and compared the values to those of GroES. From size-exclusion chromatography experiments in the presence of various concentrations of Gdn-HCl at 25 degrees C, both cpn10s showed unfolding-refolding characteristics similar to those of GroES, i.e. two-stage unfolding reactions that include formation of a partially folded monomer. Although the partially folded monomer of TmaES was considerably more stable compared to GroES and RatES, it was found that the overall stabilities of all three cpn10s were achieved significantly by inter-subunit interactions. We studied this contribution of inter-subunit interactions to overall stability in the GroES heptamer by introducing a mutation that perturbed subunit association, specifically the interaction between the two anti-parallel beta-strands at the N and C termini of this protein. From analyses of the mutants' stabilities, it was revealed that the anti-parallel beta-strands at the subunit interface are crucial for subunit association and stabilization of the heptameric GroES protein.     

LDH calcutta-1: A mutation of the B subunit of human lactate dehydrogenase

The electrophoretic variant of human LDH, Calcutta-1, occurs at phenotypic frequencies of 0–4% throughout India. The variant was examined by various electrophoretic techniques and by heat stability studies. The LD₁ (B₄) isoenzyme was purified from normal and variant bloods by affinity chromatography and ion-exchange chromatography. A minimum of five Calcutta-1 LD₁ bands was demonstrated by isoelectric focusing. Electrophoresis of variant LD₁ in high-molar urea-acrylamide denaturing gels resulted in two Calcutta-1 B subunit bands, while normal gels yielded only a single band. Homozygote Calcutta-1 LDH from red cells demonstrated a decreased heat stability, while heterozygote variant LDH showed a normal heat stability. This effect was confirmed when purified LD₁'s were compared. Evidence is presented suggesting a B-subunit variant showing thermolability in the homozygous form.The author was supported by an Australian National University Scholarship.     

植酸酶基因中稀有密码子的改造提高其在毕赤酵母中的表达量

对来源于黑曲霉N2 5(AspergillusnigerChinaStrain)的植酸酶基因phyA进行PCR介导的定点突变 ,不改变其所编码氨基酸 ,选用毕赤酵母偏爱的密码子对该基因保守序列中第 81位和第 85位的Arg密码子进行同义突变 .构建了含正确突变的克隆载体pUC18 phyAm 和酵母表达载体pPIC9k phyAm,电击转化毕赤酵母 ,经MM、MD平板筛选和产物的酶活性测定 ,筛选出突变与未突变高酶活酵母转化子各 2株 .这 4株转化子的Southern印迹结果表明 ,phyA基因以单交换方式单拷贝整合到酵母染色体DNA中 .表达产物的SDS PAGE分析表明 ,重组酵母中的植酸酶能有效分泌和表达 ,蛋白质分子大小为 70 15kD .转化子酶活测定结果表明 ,经密码子优化的突变重组酵母酶活力明显高于未进行优化的重组酵母转化子 .经密码子优化的突变重组酵母株PP NPm 8于麦芽汁培养基中诱导 36h后酶活力可达 4 76 0 0U/ml,其活力比未优化重组酵母株PP NP 2 (2 36 6 7U/ml)提高了约 1倍 ,且重组转化子遗传稳定性良好 .     

Site-directed mutagenesis and homology modeling indicate an important role of cysteine 439 in the stability of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa

Betaine aldehyde dehydrogenase (BADH) from the human pathogen Pseudomonas aeruginosa is a tetrameric enzyme that contains a catalytic Cys286 and three additional cysteine residues, Cys353, 377, and 439, per subunit. In the present study, we have investigated the role of the three non-essentials in enzyme activity and stability by homology modeling and site-directed mutagenesis. Cys353 and Cys377 are located at the protein surface with their sulfur atoms buried, while Cys439 is at the subunit interface between the monomers forming a dimeric pair. All three residues were individually mutated to alanine and Cys439 also to serine and valine. The five mutant proteins were expressed in Escherichia coli and purified to homogeneity. Their steady-state kinetics was not significantly affected, neither was their structure as indicated by circular dicroism spectropolarimetry, protein intrinsic fluorescence, and size-exclusion chromatography. However, stability was severely reduced in the Cys439 mutants particularly in C439S and C439V, which were inactive when expressed at 37 degrees C. They also exhibited higher sensitivity to thermal and chemical inactivation, and higher propensity to dissociation by dilution or exposure to low ionic strength than the wild-type enzyme. Size-exclusion chromatography indicates that substitution of Cys439 lead to unstable dimers or to stable dimeric conformations not compatible with a stable tetrameric structure. To the best of our knowledge, this is the first study of an aldehyde dehydrogenase revealing a residue at the dimer interface involved in holding the dimer, and consequently the tetramer, together.     

Impact of cysteine variants on the structure,activity, and stability of recombinant human α-galactosidase A

Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity.     

Use of Ramachandran Plot for Increasing Thermal Stability of Bacterial Formate Dehydrogenase

From analysis of Ramachandran plot for NAD+-dependent formate dehydrogenase from the methylotrophic bacterium Pseudomonas sp. 101 (FDH, EC 1.2.1.2), five amino acid residues with non-optimal values phi and psi have been located in beta- and pi-turns of the FDH polypeptide chain, e.g., Asn136, Ala191, Tyr144, Asn234, and His263. To clarify their role in the enzyme stability, the residues were replaced with Gly by means of site-directed mutagenesis. The His263Gly mutation caused FDH destabilization and a 1.3-fold increase in the monomolecular inactivation rate constant. The replacements Ala191Gly and Asn234Gly had no significant effect on the stability. The mutations Asn136Gly and Tyr144Gly resulted in higher thermal stability and decreased the inactivation rate by 1.2- and 1.4-fold, respectively. The stabilizing effect of the Tyr144Gly mutation was shown to be additive when introduced into the previously obtained mutant FDH with enhanced thermal stability.     

A single residual replacement improves the folding and stability of recombinant cassava hydroxynitrile lyase in E. coil

Substitution of Ser113 for Gly113 in the cap domain of hydroxynitrile lyase from Manihot esculenta (MeHNL) was performed by site-directed mutagenesis to improve its self-generated folding and stability under denaturation conditions. The yield of the recombinant mutant HNL1 (mut-HNL1), which had higher specific activity than the wild type HNL0 (wt-HNL0), was increased by 2 to 3-fold. Thermostability of MeHNL was also enhanced, probably due to an increase in content of the -strand secondary structure according to CD analysis. Our data in this report suggest that Ser113 significantly contributes to the in vivo folding and stability of MeHNL and demonstrates an economic advantage of mut-HNL1 over the wt-HNL0.