Post-translational modulation of sucrose-phosphate synthase from Nitrosomonas europaea

HARTMAN MD; FERRETTI MV; ARCE AL; BALLICORA MA; IGLESIAS AA; ASENCIÓN DIEZ MD; FIGUEROA CM

Mendoza

Congreso; LVIII Reunión Anual de la SAIB; 2022

SAIB

The β-proteobacteria Nitrosomonas europaea is a chemolithoautotroph that oxidizes ammonia to nitrite, which enables its biotechnological use for industrial and sewage waste treatment. Recently, much effort has been advocated for genome-based studies to decipher N. europaea biochemistry, biology, and physiology. However, our knowledge regarding the regulation of metabolic routes in response to different growing conditions remains limited. In this study, we performed a comparative analysis of the growth of Nitrosomonas under heterotrophic (2% w/v fructose) or chemolithoautotrophic (air-supplemented) conditions and evaluated their impact on sucrose metabolism by determining the levels of the putative bifunctional sucrose-phosphate synthase (SPS) protein, the main enzyme involved in sucrose synthesis. SPS has a glucosyl-transferase domain connected to a sucrose phosphatase (SPP) domain by a linker composed of 47 amino acids. Studies performed with anti-SPP antibodies revealed that the full-length SPS was only present in extracts from N. europaea grown under heterotrophic conditions. In contrast, chemolithoautotrophic cultures produced a discrete band corresponding to the SPP domain, suggesting the occurrence of a putative proteolytic event within the linker region. An in silico approach revealed that the SPS linker has a putative recognition site (RLRR, score 0.9/1) for a protease from the S8 family (PS8). Using recombinant enzymes, we confirmed in vitro the cleavage of N. europaea SPS by PS8. Moreover, recombinant SPS and a protein extract from the bacterium grown on chemolithoautotrophic conditions also showed proteolysis of the SPS substrate. Then, we studied the putative effect of the proteolytic event on SPS activity by separately producing the glucosyl-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 separated SPS-S and SPS-P proteins displayed activities that were 3- and 230-fold higher, respectively. Overall, our results pose proteolysis as a post-translational modification that could enhance SPS activity and thus carbon flux to sucrose metabolism. Moreover, 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 in specific growth conditions.