Surface Arginine Saturation Effect on Unfolding Reaction of Firefly Luciferase: A Thermodynamic and Kinetic Perspective

Replacement of some hydrophobic solvent‐exposed residues in Lampyris turkestanicus luciferase with arginine increases thermostability of this enzyme. Herein, thermodynamic and kinetic of unfolding reactions of wild type (WT), E354R/356R, E354R/356R‐I232R and E354R/356R‐Q35R/L182R/I232R variants, has been investigated. Fluorescence and Far‐UV circular dichroism measurements using urea as a chemical denaturant indicated that the value of for all variants is greater than that of WT enzyme. Analysis of m‐values, as a measure of difference in the solvent accessible surface area between the native and denatured states of protein, revealed that higher stability of mutants is related to their higher degree of compactness in the folded state. Results of unfolding kinetic experiments showed that all variants have three‐exponential behavior in which they unfolded with three rate constants and corresponding amplitudes. Increasing the rate constants of fast unfolding phase in mutants relative to WT protein may be attributed to more compactness and more kinetic sensitivity of their folded state to urea. However, more population of WT protein was unfolded from fast unfolding phase. Results of this investigation highlight kinetic stability of luciferase via a slow rate of unfolding.     

Rational Design of Catechol-2, 3-dioxygenase for Improving the Enzyme Characteristics

Catechol-2, 3-dioxygenase (C23O) from Pseudomonas sp. CGMCC2953 identified in our laboratory, which is one of the key enzymes responsible for phenanthrene biodegradation, was expected to get better characteristics tolerant to environment for its further application. With the aim of improving the enzyme properties by introducing intermolecular disulfide bonds, X-ray structure of a C23O from Pseudomonas putida MT-2, a highly conserved homologous with the C23O from Pseudomonas sp. CGMCC2953, was directly used to find the potential sites for forming disulfide bonds between two monomers of the target C23O. Two sites, Ala229 and His294, were identified and mutated to cysteine, respectively, by using site mutagenesis. The expected disulfide bond between these two CYS residues was confirmed with both molecular modeling and experimental results. The optimum temperature of the mutated enzyme was widened from 40 to 40∼50 °C. The mutated C23O became more alkalescency stable compared with the wild-type enzyme, e.g., 75% of the maximal enzyme activity retained even under pH 9.5 while 50% residue for the wild-type one. Improvement of thermostability of the mutated C230 with the redesigned disulfide was also confirmed.     

Expanding the utility of proteases in synthesis: broadening the substrate acceptance in non-coded amide bond formation using chemically modified mutants of subtilisin

The strategy of combined site directed mutagenesis and chemical modification creates chemically modified mutants (CMMs) with greatly broadened substrate specificities. We have previously reported that the CMMs of subtilisin Bacillus lentus (SBL) are efficient catalysts for the coupling of both l- and d-amino acids. We now report that these powerful catalysts also allow amide bond formation between a variety of non-coded carboxylic acids, including β-alanine and β-amino homologues of phenylalanine, with both l- and d-amino acid nucleophiles. As a guide to enzyme efficiency, a hydrolysis assay indicating pH change has been employed. CMMs selected by this screen furnished higher yields of coupling products compared to the wild-type enzyme (WT). Furthermore, both WT and CMM enzymes allow highly stereoselective aminolysis of a meso diester with an amino acid amine. These results highlight the utility of CMMs in the efficient formation of non-coded amides as potential peptide isosteres.     

Chemical- and Thermal-Induced Unfolding of Leishmania donovani Ribose-5-Phosphate Isomerase B: a Single-Tryptophan Protein

Ribose-5-phosphate isomerase B (RpiB), a crucial enzyme of pentose phosphate pathway, was proposed to be a potential drug target for visceral leishmaniasis. In this study, we have analyzed the biophysical properties of Leishmania donovani RpiB (LdRpiB) enzyme to gain insight into its unfolding pathway under various chemical and thermal denaturation conditions by using fluorescence and CD spectroscopy. LdRpiB inactivation precedes the structural transition at lower concentrations of both urea and guanidine hydrochloride (GdHCl). 8-Anilinonapthalene 1-sulfonic (ANS) binding experiments revealed the presence of molten globule intermediate at 1.5 M GdHCl and a nonnative intermediate state at 6-M urea concentration. Acrylamide quenching experiments further validated the above findings, as solvent accessibility of tryptophan residues increased with increase in GdHCl and urea concentration. The recombinant LdRpiB was completely unfolded at 6 M GdHCl, whereas the enzyme molecule was resistant to complete unfolding even at 8-M urea concentration. The GdHCl- and urea-mediated unfolding involves a three-state transition process. Thermal-induced denaturation revealed complete loss of enzyme activity at 65 °C with only 20 % secondary structure loss. The formation of the well-ordered β-sheet structures of amyloid fibrils was observed after 55 °C which increased linearly till 85 °C as detected by thioflavin T dye. This study depicts the stability of the enzyme in the presence of chemical and thermal denaturants and stability-activity relationship of the enzyme. The presence of the intermediate states may have major implications in the way the enzyme binds to its natural ligand under various conditions. Also, the present study provides insights into the properties of intermediate entities of this important enzyme.     

Cross-Linked Enzyme Aggregates of Phenylalanine Ammonia Lyase: Novel Biocatalysts for Synthesis of L-Phenylalanine

Cross-linked enzyme aggregates of phenylalanine ammonia lyase (PAL-CLEAs) from Rhodotorula glutinis were prepared. The effects of the type of aggregating agent, its concentration, and that of cross-linking agent were studied. PAL-CLEAs production was most effective using ammonium sulfate (40?% saturation), followed by cross-linking for 1?h with 0.2?% (v/v) glutaraldehyde. Moreover, the storage and operational stability of the resulting PAL-CLEAs were also investigated. Compared to the free enzyme, the PAL-CLEAs exhibited the expected increased stability of the enzyme against various deactivating conditions such as pH, temperature, denaturants, and organic solvents and showed higher storage stability than its soluble counterpart. Additionally, the reusability of PAL-CLEAs with respect to the biotransformation of l-phenylalanine was evaluated. PAL-CLEAs could be recycled at least for 12 consecutive batch reactions without dramatic activity loss, which should dramatically increase the commercial potential of PAL for synthesis of l-phenylalanine. To the best of our knowledge, this is the first report of immobilization of PAL as cross-linked enzyme aggregates.     

A Simple Technique of Preparing Stable CLEAs of Phenylalanine Ammonia Lyase Using Co-aggregation with Starch and Bovine Serum Albumin

Cross-linked enzyme aggregates (CLEAs) have been recently proposed as an alternative to conventional immobilization methods on solid carriers. However, the low cross-linking efficiency causes the major activity loss and instability in the conventional protocol for CLEA preparation. Herein, the effects of bovine serum albumin and starch addition on the cross-linking efficiency of CLEAs of phenylalanine ammonia lyase (PAL) from Rhodotorula glutinis were evaluated. A co-aggregation strategy was developed to improve cross-linking efficiency by adding starch and bovine serum albumin (BSA). CLEAs of PAL prepared in the presence of BSA and starch (PSB-CLEAs) retained 36 % activity, whereas CLEAs prepared without BSA and starch (PAL-CLEAs) retained only 8 % activity of the starting enzyme preparation. Compared with PAL-CLEAs, the thermal stability of PSB-CLEAs has improved considerably, maintaining 30 % residual activity after 4 h of incubation at 70 °C, whereas the PAL-CLEAs have only 13 % residual activity. PSB-CLEAs also exhibited the expected increased stability of PAL against hydrophilic organic solvents, superior operability, and higher storage stability. The proposed technique of preparing CLEAs using co-aggregation with starch and BSA would rank among the potential strategies for efficiently preparing robust and highly stable enzyme aggregates.     

Improvement of Drosophila acetylcholinesterase stability by elimination of a free cysteine

Background

Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use for residue detection with biosensors. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, it is not sufficiently stable for extensive utilization. It is a homodimer in which both subunits contain 8 cysteine residues. Six are involved in conserved intramolecular disulfide bridges and one is involved in an interchain disulfide bridge. The 8^th cysteine is not conserved and is present at position 290 as a free thiol pointing toward the center of the protein.

Results

The free cysteine has been mutated to valine and the resulting protein has been assayed for stability using various denaturing agents: temperature, urea, acetonitrile, freezing, proteases and spontaneous-denaturation at room temperature. It was found that the C290V mutation rendered the protein 1.1 to 2.7 fold more stable depending on the denaturing agent.

Conclusion

It seems that stabilization resulting from the cysteine to valine mutation originates from a decrease of thiol-disulfide interchanges and from an increase in the hydrophobicity of the buried side chain.     

A novel ring opening reaction of peptide N-terminal thiazolidine with 2,2′-dipyridyl disulfide (DPDS) efficient for protein chemical synthesis

In the protein chemical synthesis via native chemical ligation (NCL) method with three peptide segments, the N-terminal cysteine residue of middle segment is generally protected by thiazolidine ring. In this paper, we show the novel method for thiazolidine ring opening using 2,2′-dipyridyl disulfide (DPDS). The N-terminal thiazolidine was converted into S-pyridylsulfenylated cysteine residue with DPDS under acidic conditions, and this N-terminally Cys peptide protected with disulfide was applicable to NCL reaction without purification and deprotection steps. DPDS treatment did not remove other Cys protecting groups generally used for regioselective disulfide bond formation reactions. These results indicate that this thiazolidine ring opening reaction is quite useful for the protein chemical synthesis with three-segment NCL strategy.     

Rational Design of Practically Important Enzymes

Majority of native enzymes are poorly applicable for practical usage: that is why different methods of enzyme modification are used to obtain the biocatalysts with appropriate characteristics. Development of genome sequencing and various modern approaches in protein engineering allow one to identify protein of interest and to improve the enzyme properties for a particular process. This review describes the results on development of novel biocatalysts based on bioinformatics and rational design. New genes encoding formate dehydrogenase (FDH) from bacterium Staphylococcus aureus, yeasts Ogataea parapolymorpha and Saccharomyces cerevisiae and moss Physcomitrella patens (SauFDH, OpaFDH, SceFDH and PpaFDH, respectively), have been cloned. New FDHs were produced in the active form and characterized. SauFDH was shown to have at least 2-fold higher catalytic constant than other known FDHs. OpaFDH has catalytic parameters as good as those for soy FDH mutant forms, and in addition, is more thermostable. Apo- and holo-forms of SauFDH have been crystallized. Mutation of two Cys residues in Pseudomonas sp.101 enzyme (PseFDH) yields enzyme preparations with improved kinetic parameters and enhanced thermal and chemical stability. New generation of PseFDH preparations with the coenzyme specificity changed from NAD⁺ to NADP⁺ have been obtained. The effect of ionic liquids on the catalytic properties and thermal stability of six wild-type recombinant FDHs, and a number of their mutants, have been studied. In case of D-amino acid oxidase (DAAO), single-point mutations have been combined to create multi-point mutants. The introduced amino acid replacements have been shown to exert an additive effect, improving both kinetic parameters and increasing thermal and chemical stability. DAAO genes from Hansenula polymorpha yeast have been cloned. α-Amino acid ester hydrolase (AEH) gene has been cloned and expressed in the active form in E. coli. Structural modeling has been performed and the effectiveness in amino beta-lactams synthesis studied. The structure of a single-chain penicillin acylase from Alcaligenes faecalis (scAfPA) has been modeled and two variants of scAfPA gene was generated by PCR. Both variants have been expressed in E. coli, isolated and characterized. Catalytic properties of scAfPA were slightly better than those of its natural heterodimer.     

Folding Studies of Arginine Kinase from Euphausia superba Using Denaturants

Arginine kinase (AK) is a key metabolic enzyme for maintaining energy balance in invertebrates and studies on AK from Euphausia superba might provide important insights into the metabolic enzymes in extreme climatic marine environments. A folding study of the AK from E. superba (ESAK) has not yet been reported. To gain insights into the structural and folding mechanisms of ESAK, the denaturants guanidine HCl and urea were applied in this study. We purified ESAK from the muscle of E. superba and evaluated the inhibition kinetics with structural unfolding studies under various conditions. The results revealed that ESAK was almost completely inactivated when using 1.0 M guanidine HCl and 8.25 M urea. The kinetics, characterized via time-interval measurements, showed that the inactivations by guanidine HCl and urea were first-order reactions, with the kinetic processes shifting from monophases to biphases as concentrations increased. Measurements of intrinsic and ANS (anilinonaphthalene-8-sulfonate)-binding fluorescences showed that guanidine HCl and urea induced conspicuous changes in tertiary structures and followed the regular unfolding mechanisms. Our study provides information regarding the folding of this muscle-derived metabolic enzyme and expands our knowledge and understanding of invertebrate metabolisms.     

Does Electron Capture Dissociation Cleave Protein Disulfide Bonds?

Peptide and protein characterization by mass spectrometry (MS) relies on their dissociation in the gas phase into specific fragments whose mass values can be aligned as ‘mass ladders’ to provide sequence information and to localize possible posttranslational modifications. The most common dissociation method involves slow heating of even-electron (M+n H)ⁿ⁺ ions from electrospray ionization by energetic collisions with inert gas, and cleavage of amide backbone bonds. More recently, dissociation methods based on electron capture or transfer were found to provide far more extensive sequence coverage through unselective cleavage of backbone N–Cα bonds. As another important feature of electron capture dissociation (ECD) and electron transfer dissociation (ETD), their unique unimolecular radical ion chemistry generally preserves labile posttranslational modifications such as glycosylation and phosphorylation. Moreover, it was postulated that disulfide bond cleavage is preferred over backbone cleavage, and that capture of a single electron can break both a backbone and a disulfide bond, or even two disulfide bonds between two peptide chains. However, the proposal of preferential disulfide bond cleavage in ECD or ETD has recently been debated. The experimental data presented here reveal that the mechanism of protein disulfide bond cleavage is much more intricate than previously anticipated.     

In-Silico analysis of missense SNPs in Human HPPD gene associated with Tyrosinemia type III and Hawkinsinuria

HPPD gene codes a dioxygenase enzyme involved in catalysis of different molecules such as tyrosine and phenylalanine by oxidizing them to produce energy. A single change in protein can trigger serious genetic disorders like Tyrosinemia type III and Hawkinsinuria. This study aims to identify the functional missense SNPs of the HPPD gene by using multiple computational tools. All deleterious missense SNPs retrieved from Ensembl and OMIM database were evaluated through six different software. Ultimately, out of 148 missense SNPs, only 27 were confirmed as diseasecausing SNPs by developing a consensus approach. These damaging SNPs were further examined to evaluate their impact on protein stability and energy including their evolutionary conservation. Native and mutated proteins structures were also designed and superimposed by I-TASSER and PyMol respectively. This work results in narrowing down missense SNPs which are still not confirmed experimentally and demands the confirmation by GWAS data. Thus, these missense SNPs could directly or indirectly destabilize the amino acid interactions causing functional deviations of protein.     

The predicted unfolding order of the β-strands in the starch binding domain from Aspergillus niger glucoamylase

The unfolding of the β-strands in the starch binding domain from Aspergillus niger glucoamylase was predicted to follow the order of β3→β2→β6→β5→β4→β1→β7 by 600 ps molecular dynamics simulations at 300, 400, and 600 K. The interior region around β-strands 2 and 3 acts as the initiation site for unfolding. β-Strands 1 and 7 are probably stabilized by the disulfide bond formed between Cys509 and Cys604. β-Strand 4 is stabilized by forming an antiparallel β-sheet with β-strand 1. Hydrophobic and electrostatic interactions between side chains instead of the hydrogen bonds are important in stabilizing these β-strands, thus the entire starch binding domain.     

Characterization of an immobilized antibody-enzyme complex

Antiserum specific for propanediol dehydrogenase, an enzyme found inNeisseria gonorrhoeae cells, has been immobilized to glass. When mixed withN gonorrhoeae cell lysates, the immobilized antibody (IMA) binds the enzyme. Over 70% of the calculated adsorbed activity can be recovered from the immobilized antibody-enzyme (IMA-E) complex. When mixed with bacterial lysates prepared from different organisms having propanediol dehydrogenase-like activity, the IMA specifically adsorbed the enzyme from theN gonorrhoeae lysate. IMA-E complexes have been prepared and their kinetic, temperature and chemical stability, and antigenic properties investigated. These studies demonstrated the feasibility of using an immobilized antibody in the detection of the propanediol dehydrogenase enzyme.     

Intra- and intermolecular hydrogen bonding in formohydroxamic acid complexes with water and ammonia: infrared matrix isolation and theoretical study

The complexes of formohydroxamic acid with water and ammonia have been studied using FTIR matrix isolation spectroscopy and MP2 calculations with a 6-311++G(2d,2p) basis set. The analysis of the experimental spectra of the HCONHOH/H₂O(NH₃)/Ar matrixes indicates formation of strongly hydrogen-bonded complexes in which the NH group of formohydroxamic acid acts as a proton donor toward the oxygen atom of water or the nitrogen atom of ammonia. The NH stretching vibration of formohydroxamic acid exhibits 150 cm⁻¹ red shift in the complex with water and 443 cm⁻¹ red shift in the complex with ammonia as compared to the NH stretch of the HCONHOH monomer. The theoretical calculations indicate stability of five isomers for the water complex and three isomers for the ammonia complex. The most stable are the cyclic structures in which the water or ammonia molecules are inserted within the intramolecular hydrogen bond of the formohydroxamic acid molecule and act as proton donors for the CO group and proton acceptors for the OH group of the formohydroxamic acid molecule. In spite of their stability the cyclic structures have not been observed in the matrixes which indicates high energy barrier for their formation, the reaction of complex formation is under kinetic and not thermodynamic control.     

diSBPred: A machine learning based approach for disulfide bond prediction

The protein disulfide bond is a covalent bond that forms during post-translational modification by the oxidation of a pair of cysteines. In protein, the disulfide bond is the most frequent covalent link between amino acids after the peptide bond. It plays a significant role in three-dimensional (3D) ab initio protein structure prediction (aiPSP), stabilizing protein conformation, post-translational modification, and protein folding. In aiPSP, the location of disulfide bonds can strongly reduce the conformational space searching by imposing geometrical constraints. Existing experimental techniques for the determination of disulfide bonds are time-consuming and expensive. Thus, developing sequence-based computational methods for disulfide bond prediction becomes indispensable. This study proposed a stacking-based machine learning approach for disulfide bond prediction (diSBPred). Various useful sequence and structure-based features are extracted for effective training, including conservation profile, residue solvent accessibility, torsion angle flexibility, disorder probability, a sequential distance between cysteines, and more. The prediction of disulfide bonds is carried out in two stages: first, individual cysteines are predicted as either bonding or non-bonding; second, the cysteine-pairs are predicted as either bonding or non-bonding by including the results from cysteine bonding prediction as a feature.The examination of the relevance of the features employed in this study and the features utilized in the existing nearest neighbor algorithm (NNA) method shows that the features used in this study improve about 7.39 % in jackknife validation balanced accuracy. Moreover, for individual cysteine bonding prediction and cysteine-pair bonding prediction, diSBPred provides a 10-fold cross-validation balanced accuracy of 82.29 % and 94.20 %, respectively. Altogether, our predictor achieves an improvement of 43.25 % based on balanced accuracy compared to the existing NNA based approach. Thus, diSBPred can be utilized to annotate the cysteine bonding residues of protein sequences whose structures are unknown as well as improve the accuracy of the aiPSP method, which can further aid in experimental studies of the disulfide bond and structure determination.     

Enzyme and inhibition assay of urease by continuous monitoring of the ammonium formation based on capillary electrophoresis

We present here an easy‐to‐operate and efficient method for enzyme and inhibition assays of urease, which is a widely distributed and important enzyme that catalyzes the hydrolysis of urea to ammonia and CO₂. The assay was achieved by integrating CE technique and rapid on‐line derivatization method, allowing us to continuously drive the sample to the capillary, thus to measure the amount of the product ammonia from the beginning to the end of the reaction. The method exhibits excellent repeatability with RSD as low as 2.5% for the initial reaction rate (n = 5), with the LOD of ammonia of 20 μM (S/N = 5). The enzyme activity as well as the inhibition of urease by Cu²⁺ were investigated using the present method. The results show that Cu²⁺ is a noncompetitive inhibitor on urease, in accordance with the result published in the literature. The enzyme activity and inhibition kinetic constants were obtained and were found to be consistent with the results of traditional off‐line enzyme assays. Our study indicates that the present approach is a reliable and convenient method for analysis of the urease activity and inhibition kinetics by continuous on‐line monitoring of the ammonium formation based on CE.     

Study on the interaction mechanism between aromatic amino acids and quercetin

In this paper, we selected quercetin and aromatic amino acids (tryptophan, tyrosine, phenylalanine) as the research objects to investigate the change rules in the reaction process. The thermodynamic functions (Ka, ΔG, and ΔS) of the interactions between quercetin and aromatic amino acids (tryptophan, tyrosine, phenylalanine) were measured by isothermal titration calorimetry. The values of binding constant (Ka) reached maximum at 25°C; the entropies and Gibbs free energies were both negative at different temperatures. The kinetic parameters of quercetin and amino acids in the interaction process was determined by microcalorimetry. The results inferred that the driving force of the reaction was hydrogen bond or van der Waals force.     

Chemical Synthesis of Human Insulin‐Like Peptide‐6

Human insulin‐like peptide‐6 (INSL‐6) belongs to the insulin superfamily and shares the distinctive disulfide bond configuration of human insulin. In this report we present the first chemical synthesis of INSL‐6 utilizing fluorenylmethyloxycarbonyl‐based (Fmoc) solid‐phase peptide chemistry and regioselective disulfide bond construction protocols. Due to the presence of an oxidation‐sensitive tryptophan residue, two new orthogonal synthetic methodologies were developed. The first method involved the identification of an additive to suppress the oxidation of tryptophan during iodine‐mediated S‐acetamidomethyl (Acm) deprotection and the second utilized iodine‐free, sulfoxide‐directed disulfide bond formation. The methodologies presented here offer an efficient synthetic route to INSL‐6 and will further improve synthetic access to other multiple‐disulfide‐containing peptides with oxidation‐sensitive residues.     

Dmab/ivDde protected diaminodiacids for solid-phase synthesis of peptide disulfide-bond mimics

The use of pre-prepared diaminodiacids has been established as an effective approach for the chemical synthesis of peptide disulfide bond mimics. A technical problem often encountered in the implementation of the diaminodiacids strategy is the use of heavy metal reagents to remove the side-chain protecting groups. In the present work, we reported the development of diaminodiacid that carry 4-(N-[1-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-3-methylbutyl]amino)benzyl (Dmab) and 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde) protecting groups. This pair of protecting groups can be readily removed by mild hydrazinolysis during the solid-phase synthesis on resin. We demonstrated the use of Dmab/ivDde protected diaminodiacids in the successful synthesis of a disulfide surrogate of oxytocin.