4.A.6 The PTS Mannose-Fructose-Sorbose (Man) Family

The Man (PTS splinter group) family is unique in several respects among PTS porter families. (1) It is the only PTS family in which members possess a IID protein; (2) It is the only PTS family in which the IIB constituent is phosphorylated on a histidyl rather than a cysteyl residue. (3) Its porter members usually exhibit broad specificity for a range of sugars, rather than being specific for just one or a few sugars. The mannose porter of E. coli, for example, can transport and phosphorylate glucose, mannose, fructose, glucosamine, N-acetylglucosamine, and N-acteylmannosamine (Plumbridge and Vimr, 1999). In addition to being a transporter and the lambda receptor for transport of its DNA across the cytoplasmic membrane it is a target receptors for class IIa, IId, and IIe bacteriocins.

The structure of the E. coli IIA^Man domain has been shown to exhibit an α/β doubly wound superfold (Hu et al. 2008). The IIB domain also exhibits an α/β doubly wound superfold, but it is very dissimilar from that of the IIA domain (Orriss et al. 2003). Instead, it has the same topology as phosphoglyceromutase. Since both proteins (IIB^Man and PGM) catalyze phosphoryl transfer with a phosphohistidine intermediate, both proteins show a similar distribution of active site residues, and both exhibit similar structures, they are probably homologous. 

Solution structures of complexes between the isolated IIA^Man and IIB^Man domains of the E. coli mannose EII complex have been solved by NMR (Hu et al. 2008). The complex of wild-type IIA^Man and IIB^Man is a mixture of two species comprising a productive, phosphoryl transfer competent complex and a non-productive complex with the two active site histidines, His-10 of IIA^Man and His-175 of IIB^Man, separated by approximately 25Å. Mutation of His-10 to a glutamate to mimic phosphorylation, results in the formation of a single productive complex. The apparent equilibrium dissociation constants for the binding of both wild-type and H10E IIA^Man to IIB^Man are approximately the same (K_D ~0.5 mM). The productive complex can readily accommodate a transition state involving a pentacoordinate phosphoryl group with trigonal bipyramidal geometry bonded to the N-ε2 atom of His-10 and the N-δ1 atom of His-175 with negligible (<0.2 Å) local backbone conformational changes in the immediate vicinity of the active site. The non-productive complex is related to the productive one by an approximately 90 degree rotation and an approximately 37 Å translation of IIB^Man relative to IIA^Man, leaving the active site His-175 fully exposed to solvent in the non-productive complex (Hu et al. 2008).

The cryo EM structure of the mannose Enzyme IICD complex (ManY/ManZ, respectively) has been solved to 3.52 Å resolution (Liu et al. 2019). The structure in an inward-facing conformation,reveals a three-fold symmetry axis perpendicular to the membrane. The trimer has dimensions of ~104 Å × 104 Å × 73 Å. Each protomer is composed of a ManY and ManZ, which have similar folds and are related to each other by a pseudosymmetry axis parallel to the membrane. ManY consists of nine TMSs, 1–9Y and one horizontal periplasmic amphipathic α-helix (AH1Y), with N- and C-termini of the protein on periplasmic and cytoplasmic sides, respectively. ManZ also contains nine TMSs, but two instead of one horizontal amphipathic α-helices (AH1Z and AH2Z), with N- and C-termini of the protein on cytoplasmic and periplasmic sides, respectively. However, TMSs 1–6Z are located on the cytoplasmic side of the membrane. ManYZ oligomerization is mediated by extensive interactions between two C-terminal TMSs (TMS8Y and TMS9Y) of ManY, mostly through hydrophobic residues. ManY and ManZ can be classified as CoreY, ArmY, and VmotifY as well as CoreZ, ArmZ, and VmotifZ domains, respectively. VmotifY and VmotifZ interlock to form the Vmotif domain of the complex. CoreY and CoreZ clamp the substrate, forming the Core domain. The helices AH1Y and AH1Z are designated the ArmY and ArmZ domains. The two dissimilar subunits can be topologically superimposed, but once ManY and ManZ are aligned according to their CoreY and CoreZ domains, VmotifY and VmotifZ domains swing apart due to different orientations of ArmY and ArmZ. When ManY and ManZ are aligned according to the Vmotif, the Core domains rotate in the membrane, which is assumed to be the root of the elevator mechanism of transport. 

The structure shows a mannose molecule bound to each protomer, caged in an elipsoidal binding pocket of the core domain. The two loops, L12Y and L34Y, of ManY shape the top and left side of the cleft, whereas loops L12Z and L34Z of ManZ shape the bottom side of the pocket. The right-side wall is mainly constructed of residues from TMS5Z. The C6-hydroxyl of the substrate can be phosphorylated by IIB, and it orients to the solvent ready for this phosphorylation event (Liu et al. 2019). The structure and mechanism of mannose-type PTS Enzyme II complexes have been reviewed in detail (Jeckelmann and Erni 2020).

Transport via ManYZ may involve four sequential steps. The default state is probably an outward open state (modeled according to the pseudosymmetry between ManY and ManZ). In this state, the CoreZ domain approaches the VmotifZ domain. Then, the binding of the substrate to the pocket of the Core domain causes a switch to an inward-facing state through the movement of the Core relative to Vmotif. In this inward-facing state, CoreY is close to the VmotifY domain, and the substrate pocket is accessible from the cytoplasmic side. In the third step, IIB transfers the phosphory group from IIB~P to mannose. Mannose-6-P then leaves the binding site and enter the cytosol. Finally, using the energy coupled with the phosphate originally transferred from PEP, the Core domain returns to the default state, and the whole system restarts this cycle of transport (Liu et al. 2019). The EM DataBank # is EMD-9906 while the PDB # is 6K1H.

The generalized reaction catalyzed by members of the Man Family is:

Sugar (out) + PEP (in) → Sugar-P (in) + pyruvate (in).