Generation and characterization of ChlreSEX4 mutants

TORRESI, FLORENCIA; RICORDI, MICAELA; GOMEZ-CASATI, DIEGO F.; BUSI, MARIA V.; MARTÍN, MARIANA

Bratislava

Simposio; 8th Symposium on the Alpha-Amylase Family; 2022

Generation and characterization of ChlreSEX4 mutantsFlorencia Torresi1,2, Micaela Ricordi1,2, Diego F. Gomez-Casati1,2, M. Victoria Busi1,2& Mariana Martín1,21Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Rosario, Santa Fe, Argentina.2Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario. Argentina.e-mail:torresi@cefobi-conicet.gov.ar; martin@cefobi-conicet.gov.arThe native starch granule appears to be a poor substrate for most amylolytic enzymes and one of the crucial steps in initiating starch degradation is its phosphorylation. The addition of phosphate groups to the polymer triggers a perturbation of the granule surface transforming the polysaccharide in a better substrate for the attack of exoamylolytic enzymes. However, the phosphate groups can also obstruct some glucan hydrolytic enzymes. Thus, it has become clear that dephosphorylation of glucans byglucan phosphatases is also essential for normal storage polysaccharide degradation in plants as well as in mammals. Arabidopsis thalianaphosphoglucan phosphatases starch excess 4 (SEX4) and like-SEX4 2 (LSF2) and human Laforin are the fundamental representatives of the atypical Dual Specificity Phosphatases (DSPs), which belong to the larger Protein Tyrosine Phosphatase superfamily. In order to understand the evolution of catalysis and regulation of these enzymes we have identified and characterized ChlreSEX4, a SEX4 homologous protein belonging to the unicellular green algaC. reinhardtii. ChlreSEX4 possesses a chloroplast targeting peptide (cTP), followed by a DSP domain, a CBM domain (CBM48), and a carboxy-terminal(CT) motif.We constructed a series of mutants in the CBM or DSP domain of ChlreSEX4by site directed mutagenesis and studied phosphatase activity of the recombinant wild type and mutant enzymes as well as their ability to bind polysaccharides. We could verifythat Cys224 is the residue responsible for the nucleophilic attack during catalysis, the Trp305 is essential for amylopectin dephosphorylation and for polysaccharides binding. Furthermore, the Tyr166 is also necessary for catalysis and substrate binding but the change of Ser261 for a Phe residue has no effect in the catalysis or amylose binding. These data confirm that ChlreSEX4, possesses a contiguous ligand –binding active site in which Cys224 and Trp305 play a critical role. Thus, ChlreSEX4, together with the pneumococcal virulence factor SpuA and SEX4 are exceptions to the general consideration that the majority of CBMs are considered to be discrete entities within a polypeptide chain.All in all, our data contribute to understanding the phosphoglucan phosphatases evolutionary processin the green lineage and their role in starch reversible phosphorylation.