From genome to function: Identification and characterization of bifunctional pyrophosphate-fructose-6-phosphate 1-phosphotransferase from Candidatus Liberibacter asiaticus

Abstract

Candidatus Liberibacter asiaticus (CLas) is an obligate intracellular pathogen that causes citrus greening disease. Prolonged host association has driven reductive evolution and functional adaptation, leaving central carbon metabolism disrupted by the absence of phosphoglucose isomerase (pgi) in glycolysis and transaldolase in the pentose phosphate pathway (PPP). Here, we characterized the bifunctional enzyme, pyrophosphate-fructose-6-phosphate 1-phosphotransferase (PFP), that connects glycolysis and PPP. PFP primarily converts D-fructose 6-phosphate (F6P) to D-fructose 1,6-bisphosphate (FBP). The proposed alternative function of PFP is the conversion of D-sedoheptulose 7-phosphate (S7P) to D-sedoheptulose 1,7-bisphosphate (SBP), and this reaction is part of the sedoheptulose 1,7-bisphosphate pathway (SBPP). To validate this, the recombinant CLas PFP was heterologously expressed, purified, and biochemically characterized. Biochemical characterization showed comparable affinity for F6P and S7P, with Km values of 66.62 {+/-} 5.4 uM and 57.5 {+/-} 2.85 uM respectively. In-silico analysis showed distinct motifs that reflect specificity for phosphoryl donors among phosphofructokinase isoforms. Biophysical characterization showed that PFP loses conformational stability above 50 degree C and at pH 11.0. The study demonstrates that CLas PFP catalyzes the conversion of S7P to SBP, validating its dual function.