Effect Of N-terminal Region On The Stabilization Of Acylpeptide Hydrolase/Esterase From Hyperthermophilic Archaeon Aeropyrum Pernix K1

Phylogenetic tree and circumstancial evidence suggest thehyperthermophiles, including archaea and bacteria, were the first life-forms tohave arisen on earth. Hyperthermophilic enzyme can therefore serve as modelsystems in understanding enzyme evolution, molecular mechanisms fro proteinthermostability, and the upper temperature limit for enzyme function. Thisknowledge can lead to the development of new and more efficient proteinengineering strategies and a wide range of biotechnological applications.Thermostability at high temperatures is an inherent property of hyperthermophilicenzymes. Enough experimental evidence (e.g., sequence, mutagenesis, structure, andthermodynamics) has been accumulated on hyperthermophilic proteins in recent yearsto try to discover new factors which are responsible for the remarkable stability ofhyperthermophilic proteins. Eventhough, it seems that no single mechanism isresponsible for the remarkable stability of hyperthermophilic proteins, the factors suchas surface salt bridge networks, solvent-exposed hydrophobic surface, and anchoring of"loose ends" (i.e., the N and C termini and loops) to the protein surface seem to beinstrumental in hyperthermophilic protein thermostability. Some researcher accountthat about half proteins in PDB bank were found to have their two terminals interactedeach other. Some experimental evidence further suggested that protein stability isinflected by terminal extention or truncation, and the stability will decrease when theinteractions between the terminal and the structure were destroyed. So, to investigatethe effect of N-terminal on the stabilization of hyperthermophilic enzyme willnot only enhance our knowledge on mechanisms of protein stabilization, butalso contribute on resional design of protein stabilization.The recombinant protein APE1547 from the thermophilic archaeonAeropyrum pernix K1 is characterized as acylpeptide hydrolase and possessesboth acylpeptide hydrolase and esterase activity. APE1547 shows extremelyhigh thermalstability and solvent resistance. Recently, the crystal structure ofAPE1547 was determined. Crystal structure analysis reveals structural homologywith members of the POP family, which contain β-propeller domain andα/β-hydrolase domain. An N-terminal extension was found to connect thehydrolase domain. The possible role of the N-terminal extension is believed toprovide stability of the circular structure of the propeller by covalently linking itto the peptidase domain. By structure superposition, we found the N-terminalextension of APE1547 anchored tightly on the terminal region by hydrophobicinteractions, hydrogen bonds and salt bridges Therefore, it is speculated thatN-terminal of APE1547 may play a significance role on the protein stability.In order to investigate the stability mechanisms of N-terminal of APE1547,we constructed mutants including N-terminal truncated mutants (21del and10del) and salt bridges disrupted mutants (D15A, R18A and D15A/R18A). Thecatalytic property and stability of the wild-type and mutants were compared indetails.The optimum temperature of wild type is 95?C, by contrast with wild-type,the optimum temperatures of mutants 21del, D15A, R18A and D15A/R18A aredecreased to 92.5?C and 77.5?C, respectively. The catalytic rate of mutant 21delis slightly higher than wild type at low temperature. The optimum pH is 8.0 forwild type and 8.5 for mutant 21del, and their substrate specificities areconsistent. The thermodynamic parameters of activity were calculated. Theentropy and enthalpy of wild type are both lower than mutants, which suggestthe molecular motion lose of wild type is higher than mutants during the ES*transition state. Structure analysis of mutant 21del indicated that the diameter ofcavity which is believe to be the route of the substrate approaching the activesite located between the two domains enlarged. The hydrogen bonds around theactive site are decreased remarkably. These results suggested that N-termianlmutate increased the flexibility of active site region.Thermal denaturation was determined by substrate activation, CD,tryptophan fluorescence, hydrophobic fluorescent probe ANS and differentialscanning calorimetry (DSC) methods. The thermal inactivation constant ofmutants 21del, 10del, D15A, R18A and D15A/R18A at 85oC is 17, 4.9, 2.6, 3.8and 1.6 times to wild type, and the free energy is decreased 8.5, 4.3, 2.9, 4.0 and1.5 kJ/mol, respectively. The inactivation process is believed to be drived byentropy. Based on the CD, tryptophan fluorescence, and hydrophobicfluorescent probe ANS detection, the inactivation was occurs on the tertiarystructure level within subtle rearrangement. As recorded by DSC, Thecalculated total enthalpic changes (∑△Hcal) are 795.3, 345.0, 522.7, 523.9 and528.1kcal/mol for WT, mutant 21del, D15A, R18A and D15A/R18A,respectively. Deconvolution of the excess heat capacity (Cp) functions revealedfive subsequent transitions for APE1547, which suggested the WT contains 5calorific stable domains, while the mutants 21del, 10del, D15A and R18Acontains 2, 3, 4 and 4, respectively. Even though, the muant D15A/R18Acontains the same number calorific stable domains compared with wild type,the stability decreased about 33.6% compared to wild type calculated by totalenthalpic changes. These results above suggested that the N-terminal ofAPE1547 may enhance the stability by increasing the numbers of calorificstable domains or by increasing the intensity of each calorific stable domains.The changes in the activity and the conformation of wild-type and mutantwere determined during unfolding by guanidine hydrochloride (Gdn-HCl).Activities of wild type and mutants 21del, 10del were completely loss at 2.6M,1M and 1.4M Gdn-HCl, respectively. The activity losing is well cooperatedwith fluorescence intensity decreasing. Three-state model and four-state modelwere used to fit the unfolding process. One intermediate was found in theunfolding process of wild type and mutants D15A, R18A and D15A/R18A,while two intermediates for mutants 21del and 10del. N-terminal truncation andsalt bridge disrupting mainly decrease the stability of local region whichcorrelated active site and α/β hydrolase domain of APE1547. The free energy ofmutants 21del, 10del, D15A, R18A and D15A/R18A were decreased about 15.5,5.3, 0.3, 3.5 and 3.9 kJ/mol. Refolding experiment indicated that the unfoldingprocess of wild type and mutants D15A, R18A and D15A/R18A is reversiableduring native to the intermediate, but for the process from intermediate tounfolding state seems to irreversible, for the activity can not be recovered. Theactivity of mutant 21del can not be recovered in all concentration of Gdn-HCl.The results suggested that the N-terminal may be play important role inAPE1547 folding in vitro.In conclusion, we provide direct evidence that N-terminal of APE1547 fromthe hyperthermophilic archaeon Aeropyrum pernix K1 plays an important rolein the protein stability. We constructed a series of mutants based on the resultsof structure alignment. Enzyme characters and unfolding process which isinduced by thermal and Gdn-HCl were mensurated with CD and fluorescencespectrum technical. Combining the results gained from enzymology, kinetics ofthermal inactivation, thermodynamics and protein structure analysis, weconfirmed that the N-terminal of APE1547 participates in forming localstabilized regions by salt bridges and hydrophobic interactions. Through thisstrategy, the two domains of APE1547 pack compactly, and the stabilityenhanced by increasing the numbers of calorific stable domains or by increasingthe intensity of each calorific stable domains. The increased local regionstability further protected the flexible α/β hydrolase against denaturants. Inaddition, evidence suggests that N-terminal has taken part in the folding processof active site. This study amplified our understanding on high stability ofmulti-domain thermophilic protein and made contribution of rational design ofprotein stabilization.