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| Author(s): |
Felipe Akihiro Melo Otsuka
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) |
| Defense date: | 2023-02-08 |
| Examining board members: |
Sandro Roberto Marana;
Luis Eduardo Soares Netto
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| Advisor: | Sandro Roberto Marana |
| Abstract | |
Enzymes that catalyze chemical reactions, whose activity is sensitive to their amino acid sequence, oligomeric state and the conditions of the medium in which they are found, e.g., temperature and ionic strength. Fundamentally, statistically discrete populations with distinct conformations influence and confer states of greater and lesser activity to the enzyme. In this thesis, the interrelations between mutations, conformations and catalytic activity are discussed in two types of enzymes. In chapter I, a βglycoside hydrolase from Spodoptera frugiperda (Sfβgly) expressed and purified in a heterologous system (E. coli) is examined, which its activity is sensitive to its oligomeric state. Furthermore, cumulative mutations in its sequence can alter its stereoselectivity against synthetic substrates. The results indicate that Sfgly in solution forms an equilibrium of state between dimer and monomer with a dissociation constant (KD) of 3.7±0.3 µM, the dimeric form being about 2.5 times more active compared to its monomeric state when separated by gel filtration. However, the kinetic dimer dissociation constant (koff) estimated at 277·10-6 s-1 suggests that to reach the monomeric state it is necessary to pass through a high activation energy barrier (Ea) of about 18.5 kcal. The explanation of this phenomenon is supported by the theory that proteins have conformational states, and in the case of Sfβgly, the dimeric and monomeric state would have different conformations that would also be associated with the enzyme activity. Mutations in the dimer interface designed to destabilize the binding between the subunits validate that the monomer activity is lower compared to the fraction containing wild-type enzyme dimers. Still regarding mutations, a variant of Sfβgly with 47 accumulated mutations in its sequence generated a monomeric enzyme capable of hydrolyzing substrate with α-glycosidic bond, a rare function among β-glycosidases. In chapter II, the theme revolves around mutations in a tyrosine kinase that confer resistance to drugs (inhibitors) for the treatment of Chronic Myeloid Leukemia (CML). The causative agent ofCML and the therapeutic target is a tyrosine kinase domain of the enzyme Abl1- KD, which is constitutively active in this disease. To understand how resistance mutations act on the different conformations of Abl1-KD, we computationally simulated with Rosetta the stabilizing effect of mutations in the residue and structure in the context of the conformation. Furthermore, by inferring Abl1-KD ancestral sequences, we verified whether the primary structure positions best known to confer resistance to inhibitors suffer some degree of evolutionary constriction. The results show that the total conformational energies of the inactive form estimated by Rosetta is 4.4 ± 2.2 REU (Rosetta Energy Units) more stable compared to the active conformation, indicating that the inactive conformation is more populated. Interestingly, mutations in positions that are known to confer resistance to CML treatment inhibitors are capable of remodeling the conformational energy of Abl1-KD, making the active conformation more stable, as in the case of mutations: M244V, G250E, Q252H, Y253H, F359VC and H396P. Regarding the individual energy of the residues, mutations G250E, Q252H, Y253H, E255V, V299L, F359(C,V), and H396P are more stable in the active conformation of Abl1-KD. Finally, the evolutionary history of Abl1-KD inferred by phylogeny indicates that positions known to generate resistance have a lower rate of residue change via weight scoreposition matrix (PSSM) analysis. In conclusion, mutations in Abl1-KD capable of remodeling its conformational energy stabilizing its active form, occur in moderately conserved positions that are responsible for stabilizing the inactive form of the enzyme, and are involved in the resistance phenotype observed after abrupt selective pressure imposed by inhibitors of kinases. (AU) | |
| FAPESP's process: | 18/18537-4 - Correlation of the quaternary structure and enzyme activity in the beta-glucosidase of Spodoptera frugiperda (Sfbgly) |
| Grantee: | Felipe Akihiro Melo Otsuka |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |