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.     

Expression and characterization of mutant forms of penicillin acylase from Alcaligenes faecalis

The sequencing of six plasmids carrying a gene of penicillin acylase from Alcaligenes faecalis VKM B1518 (AfPA) revealed the presence of random mutations in the gene; they occurred during a polymerase chain reaction. Six mutant AfPAs and a wild-type enzyme were expressed in E. coli cells. The activity assay of mutant AfPAs in E. coli cells indicated that several amino acid substitutions affect the expression level of the AfPA gene and the rate of cell growth. Four mutant AfPAs were purified; their catalytic properties and thermal stability were studied. It is shown that the amino acid substitutions under study do not affect the catalytic efficiency value. Within the experimental error, the βQ133R and βK184E (the AfPA M2 mutant) substitutions had no effect on the thermal stability of the enzyme; in the case of mutants AfPA M4 (βY90H), M5 (αD132G, βR97C), and M6 (αV5E, αN183S, and βE439G), the inactivation rate constant increased 2.4, 2.75, and 8.3 times, respectively, as compared to that of the wild-type enzyme.     

Influence of Met/Leu amino acid changes on catalytic properties and oxidative and thermal stability of yeast D-amino acid oxidase

The oxidative stability of enzymes is mostly dependent on the stability of the Cys and Met residues. Three single point mutants with Met/Leu substitutions in D-amino acid oxidase (DAAO, EC 1.4.3.3) from the yeast Trigonopsis variabilis (TvDAAO) are prepared and characterized. The positions for the amino acid residue substitutions are selected based on multiple alignment of different DAAO amino acid sequences and analysis of the three-dimensional structure of TvDAAO. It is shown that the substrate specificity profile ischanged for all of the mutants. The K_M values for the small and bulky D-amino acidsare increased and decreased, respectively. One of the Met/Leu substitutions results in a two- to threefold increase in thermal stability as compared to the wild-type enzyme. A method for the determination of TvDAAO stability in the presence of hydrogen peroxide is developed and the oxidative stability of wild-type and mutant TvDAAOs is studied. It is shown that none of thethree mutations changes the oxidative stability of the enzymes.     

Preparation and characterization of multipoint yeast D-amino acid oxidase mutants with improved stability and activity

D-amino acid oxidase (DAAO) is an FAD-containing oxidoreductase that stereospecifically oxidases D-amino acids to produce α-keto-acids, an ammonium ion, and hydrogen peroxide. The most important biotechnological process involving DAAO is the production of 7-amino cephalospranic acid (7-ACA) from cephalosporin C. The reaction product, 7-ACA, is then used as a precursor for the synthesis of cephalosporin antibiotics of different generations. We previously obtained mutant DAAOs from the yeast Trigonopsis variabilis (TvDAAO). The mutants with point amino acid substitutions were characterized by either an increased thermal stability or improved catalytic properties in the oxidation of cephalosporin C. In the present study, we obtained two new mutant TvDAAOs with two and four amino acid substitutions, respectively. The catalytic constants of these mutant TvDAAOs for the oxidation of cephalosporin C were 1.8 and 4 times higher than the respective parameter of the wild-type enzyme (wt-TvDAAO). The combination of substitutions increased the thermal stabilities of both mutant TvDAAOs by a factor of 2–3 as compared with the wt- TvDAAO.     

Role of Met93 and Thr96 in the Lid Hinge Region of Rhizopus chinensis Lipase

We engineered Rhizopus chinensis lipase to study its critical amino acid role in catalytic properties. Based on the amino acid sequence and three-dimensional model of the lipase, residues located in its lid hinge region (Met93 and Thr96) were replaced with corresponding amino acid residues (Ile93 and Asn96) found in the lid hinge region of Rhizopus oryzae lipase. The substitutions in the lid hinge region affected not only substrate specificity but also the thermostability of the lipase. Both lipases preferred p-nitrophenyl laurate and glyceryl trilaurate (C12). However, the variant S4-3O showed a slight decline in activity toward long-chain fatty acid (C16–C18). When enzymes activities decreased by half, the temperature of the variant (45 °C) was 22 °C lower than the parent (67 °C), probably substantially destabilized the structure of the lid region. The interfacial kinetic analysis of S4-3O suggested that the lower catalytic efficiency was due to a higher K _m* value. According to the lipase structure investigated, Ile93Met played a role of narrowing the size of the hydrophobic patch, which affected the substrate binding affinity, and Asn96Thr destabilized the structure of the lipase by disrupting the H-bond interaction in the lid region.     

Increased thermal stability of luciferase of lighting beetles <Emphasis Type="Italic">Luciola mingrelica</Emphasis> by random mutagenesis

Luciferase of lighting bugs finds wide application in a number of fields in biotechnology and molecular biology, but use of luciferase is often limited by its fast inactivation at elevated temperatures. As a result of four sequential cycles of random mutagenesis, we obtained a mutant of luciferase of Luciola mingrelica lighting bugs with a considerably higher thermal stability. The obtained amino acid substitutions also resulted in an increase in the specific activity and a decrease in the Michaelis constant in terms of ATP by eight times, which evidences higher catalytic activity of the mutant. It is shown that using a random mutant is a highly efficient approach to increase the stability of luciferase.     

Why substituting the asparagine at position 35 in Bacillus circulans xylanase with an aspartic acid remarkably improves the enzymatic catalytic activity? A quantum chemistry-based calculation study

Xylanases from Bacillus circulans (BCX) are known as configuration-retaining glycoside hydrolases, which hydrolyze xylans with two glutamic acid residues (Glu78 and Glu172) serving as catalytic active residues according to a double displacement mechanism. Existing experimental researches show that mutating the asparagines (Asn) to aspartic acid (Asp) at position 35 next to Glu172 can obviously improve the catalytic activity of BCX. To better understand the inherent mechanism for the experimental finding, we performed quantum chemistry calculations on two model systems to mimic the catalyses of wild-type and mutant BCXs. Geometrical structures and relative energies of intermediates and transition states involved in the hydrolysis reactions are given in detail. It is found that in the wild-type model system Asn35 interacts with Glu172 via a loose hydrogen bond, while in the mutant model system Asp35 forms a very tight hydrogen bond with Glu172. The glycosidic bond cleavage is proposed to be the rate-determining step for the hydrolysis reaction, whose barrier varies from 98 to 65 kJ mol⁻¹ when Asn35 is replaced by Asp35, showing the presence of Asp35 remarkably reduces the energy demand for the hydrolysis reaction. The present result provides a theoretical elucidation for why a single amino acid substitution can importantly influences catalytic activity of BCX.     

Preparation of d-amino acids by enzymatic kinetic resolution using a mutant of penicillin-G acylase from E. coli

We have demonstrated for the first time that d-glutamine (d-Gln) and d-glutamic acid (d-Glu) can be efficiently obtained in high ee (97% and 90%, respectively) by enzymatic kinetic resolution of d,l-Gln and d,l-Glu. This was achieved by enantioselective conversion of the l-enantiomers to their N-phenylacetyl derivatives in aqueous solution, using a mutant of penicillin-G acylase (PGA) from E. coli and phenylacetic acid methylester as the acyl donor. Kinetic modeling studies suggest that the high ee values obtained are both due to a strong enantiopreference for the l-amino acid in the deacylation step of the covalent enzyme intermediate, as well as to completeness of conversion that is transiently obtained as a result of the distinct preference of the mutant PGA for phenylacetic acid methylester over the N-phenylacetyl-l-amino acid product. For the other amino acids tested (Asn, Asp, and Ser), the highest ee values that were obtained for the remaining d-enantiomer are moderate (50–80%) because of lower enantioselectivity in the enzyme deacylation step and due to less complete conversion of the l-amino acid caused by competition for the active site between the acyl donor and the N-phenylacetyl-l-amino acid that is produced. The results demonstrate that the mutated PGA has great potential for the production of optically active d-amino acids by kinetic resolution.     

Stereospecific capillary electrophoresis assays using pentapeptide substrates for the study of Aspergillus nidulans methionine sulfoxide reductase A and mutant enzymes

Stereospecific capillary electrophoresis‐based methods for the analysis of methionine sulfoxide [Met(O)]‐containing pentapeptides were developed in order to investigate the reduction of Met(O)‐containing peptide substrates by recombinant Aspergillus nidulans methionine sulfoxide reductase A (MsrA) as well as enzymes carrying mutations in position Glu99 and Asp134. The separation of the diastereomers of the N‐acetylated, C‐terminally 2,4‐dinitrophenyl (Dnp)‐labeled pentapeptides ac‐Lys‐Phe‐Met(O)‐Lys‐Lys‐Dnp, ac‐Lys‐Asp‐Met(O)‐Asn‐Lys‐Dnp and ac‐Lys‐Asn‐Met(O)‐Asp‐Lys‐Dnp was achieved in 50 mM Tris‐HCl buffers containing sulfated β‐CD in fused‐silica capillaries, while the diastereomer separation of ac‐Lys‐Asp‐Met(O)‐Asp‐Lys‐Dnp was achieved by sulfated β‐CD‐mediated MEKC. The methods were validated with regard to range, linearity, accuracy, limits of detection and quantitation as well as precision. Subsequently, the substrates were incubated with wild‐type MsrA and three mutants in the presence of dithiothreitol as reductant. Wild‐type MsrA displayed the highest activity towards all substrates compared to the mutants. Substitution of Glu99 by Gln resulted in the mutant with the lowest activity towards all substrates except for ac‐Lys‐Asn‐Met(O)‐Asp‐Lys‐Dnp, while replacement Asn for Asp134 lead to a higher activity towards ac‐Lys‐Asp‐Met(O)‐Asn‐Lys‐Dnp compared with the Glu99 mutant. The mutant with Glu instead of Asp134 was the most active among the mutant enzymes. Molecular modeling indicated that the conserved Glu99 residue is buried in the Met‐S‐(O) groove, which might contribute to the correct placing of substrates and, consequently, to the catalytic activity of MsrA, while Asp134 did not form hydrogen bonds with the substrates but only within the enzyme.     

An analysis of ATP binding with kinase catalytic subunit by molecular dynamics simulation of the CDK2 active kinase crystal lattice

Molecular dynamics simulation was used to analyze changes in the functionally significant structural elements of the crystal lattices of pT160-CDK2/cyclin and A/ATP-Mg²⁺/substrate complexes of the native (CDK2-G16) and mutant (CDK2-S16) active kinases at physiological temperatures (300 K). The structural rearrangement of ATP caused by changes in the kinase catalytic domain was studied. ATP was fixed by the ionic and H-bond interactions of several residues, including Lys33, Asp145, and side-chain amides of the G loop between β1 and β2. The binding of the kinases to complexes with cyclin and the phosphorylation of T160 in the active complex of the CDK2 kinase result in the ATP orientation more convenient for the transfer of the phosphate group to the substrate. An analysis of interatomic distances in the ATP active site region and Asp145, Asn132, Lys33 catalytic sites participating in the orientation of ATP phosphates revealed that the Asp 145 amino acid residue was situated noticeably closer to the ATP molecule in the native complex than in its mutant counterpart. The same is true of the arrangement of the Lys33 residue with respect to ATP.     

Implementing stepwise solvent elution in sequential injection chromatography for fluorimetric determination of intracellular free amino acids in the microalgae Tetraselmis gracilis

The concept of sequential injection chromatography (SIC) was exploited to automate the fluorimetric determination of amino acids after pre-column derivatization with o-phthaldialdehyde (OPA) in presence of 2-mercaptoethanol (2MCE) using a reverse phase monolithic C₁₈ stationary phase. The method is low-priced and based on five steps of isocratic elutions. The first step employs the mixture methanol: tetrahydrofuran: 10 mmol L⁻¹ phosphate buffer (pH 7.2) at the volumetric ratio of 8:1:91; the other steps use methanol: 10 mmol L⁻¹ phosphate buffer (pH 7.2) at volumetric ratios of 20:80, 35:65, 50:50 and 65:35. At a flow rate of 10 μL s⁻¹ a 25 mm long-column was able to separate aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), glycine (Gly), threonine (Thr), citruline (Ctr), arginine (Arg), alanine (Ala), tyrosine (Tyr), phenylalanine (Phe), ornithine (Orn) and lysine (Lys) with resolution >1.2 as well as methionine (Met) and valine (Val) with resolution of 0.6. Under these conditions isoleucine (Ile) and leucine (Leu) co-eluted. The entire cycle of amino acids derivatization, chromatographic separation and column conditioning at the end of separation lasted 25 min. At a flow rate of 40 μL s⁻¹ such time was reduced to 10 min at the cost of resolution worsening for the pairs Ctr/Arg and Orn/Lys. The detection limits varied from 0.092 μmol L⁻¹ for Tyr to 0.51 μmol L⁻¹ for Orn. The method was successfully applied to the determination of intracellular free amino acids in the green alga Tetraselmis gracilis during a period of seven days of cultivation. Samples spiked with known amounts of amino acids resulted in recoveries between 94 and 112%.     

Structural,Thermal, and Storage Stability of Rapana Thomasiana Hemocyanin in the Presence of Cholinium-Amino Acid-Based Ionic Liquids

Novel biocompatible compounds that stabilize proteins in solution are in demand for biomedical and/or biotechnological applications. Here, we evaluated the effect of six ionic liquids, containing mono- or dicholinium [Chol]_1or2 cation and anions of charged amino acids such as lysine [Lys], arginine [Arg], aspartic acid [Asp], or glutamic acid [Glu], on the structure, thermal, and storage stability of the Rapana thomasiana hemocyanin (RtH). RtH is a protein with huge biomedicinal potential due to its therapeutic, drug carrier, and adjuvant properties. Overall, the ionic liquids (ILs) induce changes in the secondary structure of RtH. However, the structure near the Cu-active site seems unaltered and the oxygen-binding capacity of the protein is preserved. The ILs showed weak antibacterial activity when tested against three Gram-negative and three Gram-positive bacterial strains. On the contrary, [Chol][Arg] and [Chol][Lys] exhibited high anti-biofilm activity against E. coli 25213 and S. aureus 29213 strains. In addition, the two ILs were able to protect RtH from chemical and microbiological degradation. Maintained or enhanced thermal stability of RtH was observed in the presence of all ILs tested, except for RtH-[Chol]₂[Glu].     

Determinations cinetiques par microcalorimetrie differentielle programmation de temperature. IV. Parametres cinetiques del l'homolyse de l'azo-bis-isobutyronitrile en solution

Free-radical decomposition rate constants of A.I.B.N. in different solvents, obtained from differential scanning micro-calorimetry are analyzed; calculated kinetic parameters are discussed. Free energy of activation (ΔG^≠) is proposed as a characteristic of the thermal stability of free-radical initiators.     

Mitochondria from cultured cells derived from normal and thiamine-responsive megaloblastic anemia individuals efficiently import thiamine diphosphate

Background

Within the family of green fluorescent protein (GFP) homologs, one can mark two main groups, specifically, fluorescent proteins (FPs) and non-fluorescent or chromoproteins (CPs). Structural background of differences between FPs and CPs are poorly understood to date.

Results

Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595) from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP) were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield (< 0.001). These spectral characteristics allow one to regard DsRed-NF as a true chromoprotein.

Conclusions

We located a novel point in asCP sequence (position 165) mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed.     

Interactions between Ni ions and tetrapeptides containing (Asp/Lys)HisGly(l/d)His sequence

A series of linear tetrapeptides containing two histidyl residues in positions 2 and 4 with different chirality: DHGH, DHG(d-His), KHGH, KHG(d-His), Ac-DHGH-NH₂, Ac-DHG(d-His)-NH₂, Ac-KHGH-NH₂, and Ac-KHG(d-His)-NH₂ were synthesized, characterized and their binding properties towards Ni²⁺ were investigated. To establish the stoichiometry and the stability of the resulting Ni²⁺ complexes, potentiometric titrations were carried out. The coordination mode of the complexes formed was investigated by performing extensive spectroscopic analyses (UV–Vis, CD) in strict correlation with the potentiometric results. The effects of the nature of the first amino acid (Lys versus Asp) and of the N-terminal amino group acetylation were determined. A careful comparison of the Ni²⁺ coordination abilities of the linear peptides provides a specific insight into the impact of the chirality of the C-terminal histidine residue (His⁴) on the metal binding properties.     

Structure–Activity Relationship Study of a Potent α-Thrombin Binding Aptamer Incorporating Hexitol Nucleotides

The replacement of one or more nucleotide residues in the potent α-thrombin-binding aptamer NU172 with hexitol-based nucleotides has been devised to study the effect of these substitutions on the physicochemical and functional properties of the anticoagulant agent. The incorporation of single hexitol nucleotides at the T9 and G18 positions of NU172 substantially retained the physicochemical features of the parent oligonucleotide, as a result of the biomimetic properties of the hexitol backbone. Importantly, the NU172- T ^H9 mutant exhibited a higher binding affinity toward human α-thrombin than the native aptamer and an improved stability even after 24 h in 90 % human serum, with a significant increase in the estimated half-life. The anticoagulant activity of the modified oligonucleotide was also found to be slightly preferable to NU172. Overall, these results confirm the potential of hexitol nucleotides as biomimetic agents, while laying the foundations for the development of NU172-inspired α-thrombin-binding aptamers.     

Immobilization of urease on modified styrene/polyvinylbenzene matrices

With copolymers of styrene and divinylbenzene containing various modification groups and the use of glutaric dialdehyde for their activation, immobilized forms of urease for watermelon seeds and fromStaphylococcus saprophyticus have been obtained. The properties of the immobilized preparations have been studied: the pH optimum, the temperature optimum and thermal stability, the influence of stabilizing components, kinetic features of the hydrolysis of urea, and work under column and batch conditions. The high stability of the immobilized preparations has been shown.     

A meanfield approach to the thermodynamics of a protein-solvent system with application to the oligomerization of the tumor suppressor p53

The thermodynamic stability and oligomerization status of the tumor suppressor p53 tetramerization domain have been studied experimentally and theoretically. A series of hydrophilic mutations at Met-340 and Leu-344 of human p53 were designed to disrupt the hydrophobic dimer-dimer interface of the tetrameric oligomerization domain of p53. Meanfield calculations of the free energy of the solvated mutants as a function of interdimer distance were compared with experimental data on the thermal stability and oligomeric state [tetramer, dimer, or equilibrium mixture of both] of each mutant. The calculations predicted a decreasing stability and oligomeric state for the following amino acids at residue 340: Met [tetramer] > Ser Asp, His, Gin, > Glu, Lys [dimer], whereas the experimental results showed the following order: Met [tetramer] > Ser > Gln > His, Lys > Asp, Glu [dimers]. For residue 344, the calculated trend was Leu [tetramer] > Ala > Arg, Gln, Lys [dimer], and the experimental trend was Leu [tetramer] > Ala, Arg, Gln, Lys [dimer]. The discrepancy for the lysine side chain at residue 340 is attributed to the dual nature of lysine, both hydrophobic and charged. The incorrect prediction of stability of the mutant with Asp at residue 340 is attributed to the fact that within the meanfield approach, we use the wild-type backbone configuration for all mutants, but low melting temperatures suggest a softening of the α-helices at the dimer-dimer interface. This initial application of meanfield theory toward a protein-solvent system is encouraging for the application of the theoretical model to more complex systems.     

基于复杂网络的随机森林算法预测氨基酸突变对蛋白质稳定性的影响

利用机器学习方法对单个氨基酸突变引起的蛋白质稳定性变化进行精确地预测,对蛋白质的结构和功能方面的研究具有重要的价值,并且对设计新的蛋白质及蛋白质工程学具有一定的指导意义.通过对蛋白质网络拓扑特征的研究,发现网络拓扑特征对于蛋白质突变稳定性影响具有较高的准确率.基于蛋白质网络拓扑特征的随机森林算法,能较好的对蛋白质单点突...