Modulation of sucrose-phosphate synthase from Nitrosomonas europaea

FERRETTI MV; IGLESIAS AA; ARCE A; ASENCION DIEZ MD; HARTMAN MD; BALLICORA MA; FIGUEROA CM

Rosario, Argentina

Workshop; V Encuentro y Segundo Workshop de la Red Argentina de Tecnología Enzimática (RedTEz); 2023

Red Argentina de Tecnologia Enzimatica (RedTEz)

The β-proteobacterium Nitrosomonas europaea is a chemolithoautotroph that oxidizes ammonia tonitrite, enabling its biotechnological use for industrial and sewage waste treatment. Researchperformed in recent years has been focused on genome-based studies to decipher N. europaeabiochemistry; however, our knowledge regarding the regulation of metabolic routes remains limited.In this study, we performed a comparative analysis of the growth of N. europaea under heterotrophic(2% w/v fructose) or chemolithoautotrophic (air-supplemented) conditions, and evaluated theirimpact on sucrose metabolism by determining the levels of the putative bifunctional sucrosephosphate synthase (SPS) protein, the major enzyme involved in sucrose synthesis. SPS has aglycosyl-transferase domain connected to a sucrose phosphatase (SPP) domain by a linkercomposed of 47 amino acids. Studies performed with anti-SPP antibodies revealed that the fulllength SPS was only present in extracts from N. europaea grown in heterotrophic conditions. On theother hand, chemolithoautotrophic cultures produced a discrete band corresponding to the SPPdomain, suggesting the occurrence of a proteolytic event at the level of the linker region. Indeed, insilico analyses revealed that the SPS linker possesses a putative recognition site (RLRR, score 0.9/1)for a protease from the S8 family (PS8). Using recombinant enzymes, we confirmed in vitro thecleavage of N. europaea SPS by the PS8 protease. Moreover, recombinant SPS and a protein extractfrom the bacterium grown on chemolithoautotrophic conditions also showed proteolysis of the SPS substrate. Then, we studied the effect of the proteolysis on SPS activity by separately producing the glycosyl-transferase domain (SPS-S) from the SPP domain (SPS-P). While the full-length SPS exhibited values of 0.065 and 0.012 U/mg for the synthase and the phosphatase activities, the individual SPS-S and SPS-P proteins displayed 3- and 230-fold higher activities, respectively. In a whole view, results presented here pose proteolysis as a post-translational modification that could enhance SPS activity and thus carbon flux to sucrose metabolism. In addition, we found that almost 7% of the total N. europaea proteome (~2000 proteins) has the consensus RXRR/RXKR site for SP8 recognition, which opens multiple possibilities for future research in proteome reshaping mediated by these and other proteases that are expressed under specific growth conditions.