A biophysical and structural study of two chitinases from Agave tequilana and their potential role as defense proteins
Por:
Sierra-Gomez, Yusvel, Rodriguez-Hernandez, Annia, Cano-Sanchez, Patricia, Gomez-Velasco, Homero, Hernandez-Santoyo, Alejandra, Siliqi, Dritan, Rodriguez-Romero, Adela
Publicada:
1 dic 2019
Ahead of Print:
1 jul 2019
Resumen:
Plant chitinases are enzymes that have several functions, including
providing protection against pathogens. Agave tequilana is an
economically important plant that is poorly studied. Here, we identified
a chitinase from short reads of the A. tequilana transcriptome (AtChi1).
A second chitinase, differing by only six residues from the first, was
isolated from total RNA of plants infected with Fusarium oxysporum
(AtChi2). Both enzymes were overexpressed in Escherichia coli and
analysis of their sequences indicated that they belong to the class I
glycoside hydrolase family19, whose members exhibit two domains: a
carbohydrate-binding module and a catalytic domain, connected by a
flexible linker. Activity assays and thermal shift experiments
demonstrated that the recombinant Agave enzymes are highly thermostable
acidic endochitinases with Tm values of 75 degrees C and 71 degrees C.
Both exhibit a molecular mass close to 32 kDa, as determined by
MALDI-TOF, and experimental pIs of 3.7 and 3.9. Coupling small-angle
x-ray scattering information with homology modeling and docking
simulations allowed us to structurally characterize both chitinases,
which notably show different interactions in the binding groove. Even
when the six different amino acids are all exposed to solvent in the
loops located near the linker and opposite to the binding site, they
confer distinct kinetic parameters against colloidal chitin and similar
affinity for (GlnNAc)(6,) as shown by isothermal titration calorimetry.
Interestingly, binding is more enthalpy-driven for AtChi2. Whereas the
physiological role of these chitinases remains unknown, we demonstrate
that they exhibit important antifungal activity against chitin-rich
fungi such as Aspergillus sp. Database SAXS structural data are
available in the SASBDB database with accession numbers SASDDE7 and
SASDDA6. Enzymes Chitinases ().
Filiaciones:
Sierra-Gomez, Yusvel:
Instituto de Química, Universidad Nacional Autónoma de México, Ciudad de México, Mexico
Univ Nacl Autonoma Mexico, Inst Quim, Ciudad Univ, Ciudad De Mexico 04510, Mexico
Rodriguez-Hernandez, Annia:
Instituto de Química, Universidad Nacional Autónoma de México, Ciudad de México, Mexico
Univ Nacl Autonoma Mexico, Inst Quim, Ciudad Univ, Ciudad De Mexico 04510, Mexico
Cano-Sanchez, Patricia:
Instituto de Química, Universidad Nacional Autónoma de México, Ciudad de México, Mexico
Univ Nacl Autonoma Mexico, Inst Quim, Ciudad Univ, Ciudad De Mexico 04510, Mexico
Gomez-Velasco, Homero:
Instituto de Química, Universidad Nacional Autónoma de México, Ciudad de México, Mexico
Univ Nacl Autonoma Mexico, Inst Quim, Ciudad Univ, Ciudad De Mexico 04510, Mexico
Hernandez-Santoyo, Alejandra:
Instituto de Química, Universidad Nacional Autónoma de México, Ciudad de México, Mexico
Univ Nacl Autonoma Mexico, Inst Quim, Ciudad Univ, Ciudad De Mexico 04510, Mexico
Siliqi, Dritan:
Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Bari, Italy
CNR, Ist Cristallog, Bari, Italy
Rodriguez-Romero, Adela:
Instituto de Química, Universidad Nacional Autónoma de México, Ciudad de México, Mexico
Univ Nacl Autonoma Mexico, Inst Quim, Ciudad Univ, Ciudad De Mexico 04510, Mexico
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