Modulation of sucrose-phosphate synthase from Nitrosomonas europaea

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

Rosario

Congreso; V Encuentro y II Workshop de la Red Argentina de Tecnología Enzimática; 2023

RedTEz

The β-proteobacterium Nitrosomonas europaea is achemolithoautotroph that oxidizes ammonia to nitrite, enabling itsbiotechnological use for industrial and sewage waste treatment. Researchperformed in recent years has been focused on genome-based studies to decipherN. europaea biochemistry; however, our knowledge regarding the regulation ofmetabolic routes remains limited. In this study, we performed a comparativeanalysis of the growth of N. europaea under heterotrophic (2% w/v fructose) orchemolithoautotrophic (air-supplemented) conditions, and evaluated their impacton sucrose metabolism by determining the levels of the putative bifunctionalsucrose-phosphate synthase (SPS) protein, the major enzyme involved in sucrosesynthesis. SPS has a glycosyl-transferase domain connected to a sucrosephosphatase (SPP) domain by a linker composed of 47 amino acids. Studiesperformed with anti-SPP antibodies revealed that the full-length SPS was onlypresent in extracts from N. europaea grown in heterotrophic conditions. On theother hand, chemolithoautotrophic cultures produced a discrete bandcorresponding to the SPP domain, suggesting the occurrence of a proteolyticevent at the level of the linker region. Indeed, in silico analyses revealedthat the SPS linker possesses a putative recognition site (RLRR, score 0.9/1)for a protease from the S8 family (PS8). Using recombinant enzymes, weconfirmed in vitro the cleavage of N. europaea SPS by the PS8 protease.Moreover, recombinant SPS and a protein extract from the bacterium grown onchemolithoautotrophic conditions also showed proteolysis of the SPS substrate.Then, we studied the effect of the proteolysis on SPS activity by separatelyproducing 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 thesynthase and the phosphatase activities, the individual SPS-S and SPS-Pproteins displayed 3- and 230-fold higher activities, respectively. In a wholeview, results presented here pose proteolysis as a post-translationalmodification that could enhance SPS activity and thus carbon flux to sucrosemetabolism. In addition, we found that almost 7% of the total N. europaeaproteome (~2000 proteins) has the consensus RXRR/RXKR site for SP8 recognition,which opens multiple possibilities for future research in proteome reshapingmediated by these and other proteases that are expressed under specific growthconditions.