Banca de DEFESA: Izadora Cristina Moreira de Oliveira

Uma banca de DEFESA de DOUTORADO foi cadastrada pelo programa.
STUDENT : Izadora Cristina Moreira de Oliveira
DATE: 30/01/2023
TIME: 14:00
LOCAL: Auditório 4
TITLE:

BIOCHEMICAL AND STRUCTURAL ANALYSIS OF A RECOMBINANT ENDOXYLANASE FROM Humicola grisea var. thermoidea IN COMPLEX WITH FERULIC ACID

KEY WORDS:

endoxylanase, ferulic acid, structural characterization, enzymology, protein structure, molecular docking

PAGES: 186
BIG AREA: Ciências Biológicas
AREA: Biologia Geral
SUMMARY:

An endo-1,4-beta-xylanase from Humicola grisea var. thermoidea belonging to the GH11 family was obtained by expression in Pichia pastoris and named HXYN2. This enzyme has 227 amino acids and a molecular mass of 23 kDa and is thermally stable, with greater activity in the range of 40-50 °C and pH 5.0-9.0, and optimum temperature and pH of 50 °C and 6.0, respectively. In this pH range, HXYN2 presents a stable structure with slight conformational changes, indicated by fluorescence emission spectra with slight displacement of the emission bands, consistent with tryptophan residues exposed to the solvent, and changes in the side chains of tryptophan residues and/or of amino acid residues that are in the tryptophan microenvironment. Circular dichroism (CD) spectra of the enzyme at pH 4.0, 6.0, 7.0 and 9.0 showed typical bands at wavelengths that correspond to proteins with β-sheet structures. The thermal denaturation curves obtained at pH 6.0, 7.0 and 9.0 showed two states of the protein, native and unfolded, with a melting temperature (Tm) between 50 and 60 °C. At pH 4.0, this point was identified between 65 and 70 °C, which suggests that HXYN2 is more structurally stable at acidic pH. HXYN2 activity was increased by 75% in the presence of the phenolic compound ferulic acid (FA), without altering its affinity for the beechwood xylan substrate. However, FA increased the Vmax, the catalytic efficiency and the turnover number of the enzyme. Data from ITC, fluorescence quenching and molecular docking showed that AF forms a complex with HXYN2 in the aglycone region, interacting with solvent-exposed tryptophan residues and other residues in this vicinity, through hydrogen bonds, hydrophobic and ionic interactions. Binding constant values for the HXYN2:FA complex are pH dependent and indicated moderate (at pH 7.0 and 9.0) to strong (at pH 4.0) affinity. In the presence of FA, the fluorescence spectra of HXYN2 showed pH-dependent changes in the position and intensity of the emission bands. The secondary structure of HXYN2 in the presence of FA was altered with a decrease in the α-helix and an increase in the β-turn and disordered structures. The thermal denaturation curve in the presence of FA showed a Tm of 54 °C, indicating lower structural stability of HXYN2 in the presence of this compound. HXYN2 crystals were obtained under different crystallization conditions in the screening step. The refinement of these conditions resulted in crystals of different shapes and sizes. Two of these crystals were taken to the National Laboratory of Synchrotron Light – Sirius for the collection of X-ray diffraction data. The diffraction data were processed, indicating that the space group P212121 (orthorhombic) and the presence of a dimer in the asymmetric unit. The crystallographic structure of HXYN2 was obtained by molecular replacement using the coordinates of the structure of endoxylanase from Thermomyces lanuginosus (PDB 1YNA) as a model.

BANKING MEMBERS:
Externo à Instituição - IVO DE ALMEIDA MARQUES - UFG
Interna - 2222088 - ELIANE FERREIRA NORONHA
Externa à Instituição - ERICA CRISTINA MORENO NASCIMENTO - UnB
Interna - 1207115 - ILDINETE SILVA PEREIRA
Presidente - 1122620 - SONIA MARIA DE FREITAS