Klaas M. Pos

Substrate-binding proteins (SBPs) are part of solute transport systems and serve to increase substrate affinity and uptake rates. In contrast to primary transport systems, the mechanism of SBP-dependent secondary transport is not well understood. Functional studies have thus far focused on Na + -coupled Tripartite ATP-independent periplasmic (TRAP) transporters for sialic acid. Herein, we report the in vitro functional characterization of TAXIPm-PQM from the human pathogen Proteus mirabilis . TAXIPm-PQM belongs to a TRAP-subfamily using a different type of SBP, designated TRAP-associated extracytoplasmic immunogenic (TAXI) protein. TAXIPm-PQM catalyzes proton-dependent α-ketoglutarate symport and its SBP is an essential component of the transport mechanism. Importantly, TAXIPm-PQM represents the first functionally characterized SBP-dependent secondary transporter that does not rely on a soluble SBP, but uses a membrane-anchored SBP instead.

Significance Mannitol biosynthesis is essential for Acinetobacter baumannii to cope with osmotic stress. Currently, only Pseudomonas putida , Acinetobacter baylyi , and A. baumannii are able to de novo synthesize mannitol by a structurally unique bifunctional mannitol-1-phosphate dehydrogenase/phosphatase (AbMtlD). The molecular mechanism of reduction and dephosphorylation of fructose-6-phosphate to mannitol is highly dependent on the substrate shuffling from one protomer to the other protomer by a unique helix–loop–helix domain–mediated dimer formation, thus ensuring unidirectional and efficient biosynthesis of mannitol. These observations support an evolutionary adaptation of AbMtlD by fusion of dehydrogenase and phosphatase domains to facilitate efficient unidirectional enzymatic production of mannitol, unifying regulatory control and minimizing the intracellular concentration of toxic mannitol-1-phosphate during salt stress.