| Current theory and evidence suggest that hyperthermophiles were the first life-forms to have arisen on Earth. Hyperthermophilic enzymes can therefore serve as model systems for use by biologists interested in understanding enzyme evolution, molecular mechanisms for protein thermostability, and the upper temperature limit for enzyme function. for explain the molecular determinants of extreme protein thermostability. This knowledge can lead to the development of new or more efficient protein engineering strategies and a wide range of biotechnological applications.Thermostability seems to be a property acquired by a protein through a combination of many small structural modifications that are achieved with the exchange of some amino acids for others and the modulation of the canonical forces (e.g. hydrogen bonds, ion-pair interactions, hydrophobic interactions) found in all proteins.Site-directed mutagenesis experiments and comparisons of structure and stability of thermozymes and mesozymes have revealed some important factors that contribute to the remarkable stability of thermozymes,but there is no single universal mechanism that promotes stability. Mutational studies of hydrogen bonding in proteins usually replace one member of a hydrogen bonding pair with a residue incapable of hydrogen bonding.It is clear that intramolecular hydrogen bonds are essential to the structure and stability of globular proteins.By measuring the difference in stability between the mutant and the wild-type protein, one hopes to gain insight into the contribution of hydrogen bonding to the stability of the protein.APE1547 is a hyperthermophilic enzyme showed both esterase (EC 3.1.1.1) activities and acylamino acid-releasing enzyme (AARE) (EC 3.4.19.1) activities. It is very stable and shows high activity from 70 to 100°C. The gene of APE1547 from the aerobic thermophilic Aeropyrum pernix K1 encoded 582 amino acid residues was cloned and expressed in Escherichia coli and the crystal structure of the recombinant protein resolved by X-ray crystallography showed that the enzyme exists as a symmetric homodimer, and each subunit is comprised of two domains. The C-terminal domain (residues 325–581) contains a canonicalα/βhydrolase fold, with a central eight-strand mixedβ-sheet. The N-terminal domain (residues 24–324) contains aβ-propeller with seven blades; each blade consists of a four-stranded antiparallelβ-sheet. A shortαhelix at the N-terminal (residues 8–23) extends from theβ- propeller domain and forms part of the hydrolase's domain. Between the two domains is a large cavity approximately 45 ? in width. The three-dimensional arrangement of Ser445, Asp524, and His556 in APE1547 is a conserved Ser-Asp-His catalytic triad, which located in the C-terminal hydrolase domain and is covered by the N-terminalβ- propeller domainBy sequence alignment, we found that APE1547 hyperthermophilic esterase belongs to prolyl oligopeptidase family. The structural analysis of APE1547 revealed a unique feature of this enzyme: two side-chain hydrogen bond interactions (Asp34-Arg292 and Arg287- His71) exist between the blades 1 and 7 and blades 2 and 7 of theβ- propeller domain. And there are three hydrogen bonding interactions (Thr127-Gly154, Leu182-Arg145-Glu122) between the blades 3 and 4 in the propelor domain.To examine the role of these hydrogen bonds, the mutants D34E, R287G, D34E/R287G and T127V, R145V, T127V/R145V, were created.The these hydrogen interactions were deleted. The conformational features, stability, and activity of the wild type and mutated proteins have been compared by using circular dichroism, fluorescence spectroscopy, thermoinactivation, GdnHCl-induced denaturation, and kinetic assays.The optimum temperature of wild type is 95?C, the optimum temperatures of mutants D34E, R287G, T127V and R145V are decreased to 92.5?C and double mutats D34E/R287G and T127V/R145V are decreased to 90?C. The optimum pH and their substrate specificities are consistent.The catalytic efficiency kcat/km values for the D34E, R287G and D34E/ R287G were 114.1% 106.2% and 127.5% of that of the wild-type APE1547, respectively, which suggested that the catalytic abilities for the mutants were improved.The kinetic parameters of APE1547 and mutats suggest the catalytic efficiency kcat/km values for the D34E, R287G, T127V, R145V, T127V/R145V and D34E/R287G were 114.1%, 106.2%, 107.5%, 114%, 117.3% and 127.5% of that of the wild-type APE1547, respectively, which suggested that the catalytic abilities for the mutants were improved.The removal of hydrogen bonds between blades in the mutants further decrease the weak interaction of these blades. The propeller becomes more flexible and can easily release the products than the wide type. With the improvements of releasing of products all the mutants have improved kcat/km and higher catalytic activity.The secondary structure and tertiary structure of the wild type APE1547 and mutanted proteins have been compared by using CD, tryptophan fluorescence, fluorescent probe Nile Red. The results clearly show that the double mutats D34E/R287G and T127V/R145V mutation significantly alters the secondary and tertiary structures of the protein, producing a partially unfolding state; and the three-dimension structure of the protein becomes more flexible, and the tryptophan's local surroundings have changed.In contrast, the D34E, R287G and T127V, R145V, mutation has no such effect on structure.The half-lives of mutants D34E, R287G, T127V, R145V and D34E/R287G, T127V/R145V were about 5.71, 4, 4.9, 3.9, 11.9, 10.32 hour less than the wild-type enzyme, respectively. The thermal inactivation constant of mutants D34E, R287G, D34E/R287G and T127V, R145V, T127V/R145V at 85oC is about 1.35%-4 times to wild type, and the free energy is decreased 1.38, 1.13, 4.52, 1.14, 0.88 and 3.35 kJ/mol, respectively.To study the thermodynamics of the heat-induced unfolding of the proteins, differential scanning calorimetry (DSC) were used. The Tm values for D34E, R287G, T127V, R145V and double mutants D34E/R287G, T127V/R145V decreased 5.5°C, 3.6°C, 2.2, 4.3 and 9.1℃, 11.9°C, respectively, compared to the wild-type APE1547, and The denaturation enthalpy ?Htot of the wile type emzyme was 767.2kJ/mol. The ?Htot values of mutants were ranged from 449.9 to 543.1 kcal/mol, significantly lower than wild-type APE1547.The changes in the activity and the conformation of wild-type and mutant were determined during unfolding by guanidine hydrochloride (GdnHCl). Results shows the two-domain APE1547 showed a three-state folding pattern, while a single domain enzyme lacks the intermediate state. and the final denaturation concentration of the GdnHCl were generally lower for the mutants than the wild type enzyme. The free energy of mutants D34E, R287G, T127V, R145V, D34E/R287G and T127V/R145V were decreased about 8.56, 5.36, 4.35, 6.52and 15, 10.53 kJ/mol than the wile type enzyme, respectively. These results above suggested that the N-terminal of APE1547 may enhance the stability by increasing the rigidity of calorific stable domains or by increasing the intensity of each calorific stable domains.In summary, our results demonstrate that the these hydrogen bonds of Asp34-Arg292, Arg287-His71, Thr127-Gly154, Leu182- Arg145-Glu122 play very important role on keeping the integrity of the conformation, thermostability of the enzyme; they also reduce the activity of the enzyme by constrict the local conformation. The side-chain hydrogen bonds located in these special sites of the protein can significantly influence the conformational integrity and function of the entire protein and they are thus indispensable features of the hyperthermophilic esterase APE1547.This paper provided a model for study the relationship between the structure and function of hyperthermophilic esterase.
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