AUTHOR=Shobade Samuel O. , Zabotina Olga A. , Nilsen-Hamilton Marit 

TITLE=Plant root associated chitinases: structures and functions

JOURNAL=Frontiers in Plant Science

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2024.1344142

DOI=10.3389/fpls.2024.1344142

ISSN=1664-462X

ABSTRACT=<p>Chitinases degrade chitin, a linear homopolymer of β-1,4-linked N-acetyl-D-glucosamine (GlcNAc) residues found in the cell walls of fungi and the exoskeletons of arthropods. They are secreted by the roots into the rhizosphere, a complex and dynamic environment where intense nutrient exchange occurs between plants and microbes. Here we modeled, expressed, purified, and characterized <italic>Zea mays</italic> and <italic>Oryza sativa</italic> root chitinases, and the chitinase of a symbiotic bacterium, <italic>Chitinophaga oryzae</italic> 1303 for their activities with chitin, di-, tri-, and tetra-saccharides and <italic>Aspergillus niger</italic>, with the goal of determining their role(s) in the rhizosphere and better understanding the molecular mechanisms underlying plant-microbe interactions. We show that <italic>Zea mays</italic> basic endochitinase (<italic>Zm</italic>Chi19A) and <italic>Oryza sativa</italic> chitinase (<italic>Os</italic>Chi19A) are from the GH19 chitinase family. The <italic>Chitinophaga oryzae</italic> 1303 chitinase (<italic>Csp</italic>Ch18A) belongs to the GH18 family. The three enzymes have similar apparent <italic>K</italic><sub>M</sub> values of (20-40 µM) for the substrate 4-MU-GlcNAc<sub>3</sub>. They vary in their pH and temperature optima with <italic>Os</italic>Chi19A activity optimal between pH 5–7 and 30–40°C while <italic>Zm</italic>Chi19A and <italic>Csp</italic>Ch18A activities were optimal at pH 7-9 and 50–60°C. Modeling and site-directed mutation of <italic>Zm</italic>Chi19A identified the catalytic cleft and the active residues E147 and E169 strategically positioned at ~8.6Å from each other in the folded protein. Cleavage of 4-MU-GlcNAc<sub>3</sub> was unaffected by the absence of the CBD but diminished in the absence of the flexible C-terminal domain. However, unlike for the soluble substrate, the CBD and the newly identified flexible C-terminal domain were vital for inhibiting <italic>Aspergillus niger</italic> growth. The results are consistent with the involvement of the plant chitinases in defense against pathogens like fungi that have chitin exoskeletons. In summary, we have characterized the functional features and structural domains necessary for the activity of two plant root chitinases that are believed to be involved in plant defense and a bacterial chitinase that, along with the plant chitinases, may participate in nutrient recycling in the rhizosphere.</p>