Mechanism of Phosphoryl Transfer in the Dimeric IIABMan Subunit of the Escherichia coliMannose Transporter

Abstract

The mannose transporter of bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) mediates uptake of mannose, glucose, and related hexoses by a mechanism that couples translocation with phosphorylation of the substrate. It consists of the transmembrane IIC Man ⅐IID Man complex and the cytoplasmic IIAB Man subunit. IIAB Man has two domains (IIA and IIB) that are linked by a 60-Å long alanine-proline-rich linker. IIAB Man transfers phosphoryl groups from the phospho-histidine-containing phospho-carrier protein of the PTS to His-10 on IIA, hence to His-175 on IIB, and finally to the 6-OH of the transported hexose. IIAB Man occurs as a stable homodimer. The subunit contact is mediated by a swap of ␤-strands and an extensive contact area between the IIA domains. The H10C and H175C single and the H10C/H175C double mutants were used to characterize the phosphoryl transfer between IIA to IIB. Subunits do not exchange between dimers under physiological conditions, but slow phosphoryl transfer can take place between subunits from different dimers. Heterodimers of different subunits were produced in vitro by GuHCl-induced unfolding and refolding of mixtures of two different homodimers. With respect to wild-type homodimers, the heterodimers have the following activities: wild-type⅐H10C, 50%; wild-type⅐H175C 45%; H10C⅐H175C, 37%; and wild-type⅐H10C/H175C (double mutant), 29%. Taken together, this indicates that both cis and trans pathways contribute to the maximal phosphotransferase activity of IIAB Man . A phosphoryl group on a IIA domain can be transferred either to the IIB domain on the same or on the second subunit in the dimer, and interruption of one of the two pathways results in a reduction of the activity to 70 -80% of the control.The carbohydrate transporters of the bacterial phosphotransferase system (enzymes II of the PTS) 1 mediate uptake concomitant with phosphorylation of hexoses and hexitols.They consist of four functional units termed IIA, IIB, IIC, and IID that occur either as individual subunits in a protein complex or as independently folding domains of a multidomain protein. IIA and IIB sequentially transfer a phosphoryl group from the phosphoryl carrier protein HPr to the transported substrate. IIC and IID span the membrane and mediate substrate translocation. Substrate translocation is activated by the phosphorylation/dephosphorylation cycle of IIB (1-4). IIA and IIB of certain transporters have regulatory activity in addition to their "energy-transducing" function. For instance, IIA Glc of Escherichia coli, the gene product of crr, modulates the activities of adenylate cyclase (5, 6), glycerol kinase (7), and of the membrane permeases for lactose and maltose (8 -12). The IIB domains of some PTS transporters regulate the activity of antiterminator and transcription activator proteins (13). In the absence of the cognate substrate, the IIB domain of the ␤-glucoside transporter (IIBCA Bgl ) phosphorylates the antiterminator protein BglG and thereby inactivates it. This way, IIBCA Bgl feedbac...

Crystal Structure of the Citrobacter freundii Dihydroxyacetone Kinase Reveals an Eight-stranded α-Helical Barrel ATP-binding Domain

“…2). We envisage the K-and L-domains as mobile in solution and connected only by a long flexible linker as it was shown for the dimeric IIAB Man subunit of the E. coli mannose transporter (51). Attempts to induce alternative conformations by co-crystallization instead of soaking with Dha/ANP and with the heterobifunctional substrate ATP-dihydroxyacetone 2 were unsuccessful.…”

Solution NMR Structures of Productive and Non-productive Complexes between the A and B Domains of the Cytoplasmic Subunit of the Mannose Transporter of the Escherichia coli Phosphotransferase System

“…The A (residues 1-134) and B (residues 160 -323) domains of IIAB Man are covalently attached by a flexible 25-residue alanine/proline-rich linker (residues 135-159) and fold independently of one another (15). Phosphoryl transfer occurs between His-10 of IIA Man and His-175 of IIB Man (14,17). In the homologous sorbose (II Sor ) permease of Klebsiella pneumoniae (18) and fructose (II Lev ) permease of Bacillus subtilis (19), the A and B domains are expressed as separate polypeptide chains.…”

Characterization of Soluble Enzyme II Complexes of the Escherichia coli Phosphotransferase System

“…The PTS permeases are called enzymes II, and the three best characterized such enzymes are the glucose permease (II Glc ) (4,7,20), the mannitol permease (II Mtl ) (14, 18, 21-23, 26, 45), and the mannose permease (II Man ) (8, 9, 11-13, 27, 33, 34). These enzymes exist in the membrane as oligomers, most probably as homodimers (5,12,23,28,31,36,37,39,47). They catalyze two vectorial chemical reactions, sugar phosphorylation using PEP as the phosphoryl donor, dependent on Enzyme I, HPr, and IIA as well as IIBC or IIBCD (the PEP reaction), and transphosphorylation using a sugar phosphate (glucose-6-phosphate [glucose-6-P] for II Glc and II…”