Generation and characterization of ChlreSEX4 mutants.

TORRESI, FLORENCIA; RICORDI, MICAELA; DIEGO FABIAN GOMEZ CASATI; MARTIN, M.

Smolenice

Congreso; 8th Symposium on the alpha-amylase family, ALAMY_8th; 2022

Slovak Academy of Sciences

 The native starch granule appears to be a poorsubstrate for most amylolytic enzymes and one of the crucial steps ininitiating starch degradation is its phosphorylation. The addition of phosphate groups to the polymer triggers a perturbationof the granule surface transforming the polysaccharide in a better substratefor the attack of exoamylolytic enzymes. However, the phosphate groups can alsoobstruct some glucan hydrolytic enzymes. Thus, it has become clear thatdephosphorylation of glucans by glucan phosphatases is also essential fornormal storage polysaccharide degradation in plants as well as in mammals. Arabidopsisthaliana phosphoglucan phosphatases starchexcess 4 (SEX4) and like-SEX4 2 (LSF2) and human Laforin are the fundamentalrepresentatives of the atypical Dual Specificity Phosphatases (DSPs), whichbelong to the larger Protein Tyrosine Phosphatase superfamily. In order tounderstand the evolution of catalysis and regulation of these enzymes we haveidentified and characterized ChlreSEX4,a SEX4 homologous protein belonging to the unicellular green alga C. reinhardtii. ChlreSEX4 possesses a chloroplast targeting peptide (cTP), followedby a DSP domain, a CBM domain (CBM48), and a carboxy-terminal (CT) motif. Weconstructed a series of mutants in the CBM or DSP domain of ChlreSEX4 by site directed mutagenesisand studied phosphatase activity of the recombinant wild type and mutantenzymes as well as their ability to bind polysaccharides. We could verify that Cys224is the residue responsible for the nucleophilic attack during catalysis, theTrp305 is essential for amylopectin dephosphorylation and for polysaccharidesbinding. Furthermore, the Tyr166 is also necessary for catalysis and substratebinding but the change of Ser261 for a Phe residue has no effect in thecatalysis or amylose binding. These data confirm that ChlreSEX4,possesses a contiguous ligand –binding active site in which Cys224 and Trp305play a critical role. Thus, ChlreSEX4, together with the pneumococcalvirulence factor SpuA and SEX4 are exceptions to the general consideration thatthe majority of CBMs are considered to be discrete entities within apolypeptide chain. These results contribute to understand the phosphoglucanphosphatases evolutionary process in the green lineage and their role in starchreversible phosphorylation.