The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) 2 comprises a fundamental signal transduction pathway whereby phosphotransfer via a series of biomolecular steps is coupled to sugar transport across the membrane (1-4). The phosphoryl group originates on phosphoenolpyruvate, and the initial transfer steps, involving first enzyme I and subsequently HPr, are common to all components of the pathway. Thereafter, the phosphoryl group is transferred to sugar-specific enzymes II, of which there are four major classes, glucose (Glc), mannitol (Mtl), mannose (Man), and chitobiose (Chb), which share no sequence similarity with one another (2, 3) and, with one exception (the B domains of enzymes II Mtl and II Chb ), no structural similarity with one another either (4 -15). Enzymes II generally comprise three domains, two cytoplasmic, A and B, and one transmembrane, C, which may or may not be covalently linked to one another (2-4). The A domain accepts the phosphoryl group from HPr and donates it to the B domain. Subsequently, the phosphoryl group is transferred from the B domain to the incoming sugar bound to the C domain. ) of the mannitol transporter, residues 490 -637, was cloned as described previously (18). From this original construct, the active site histidine residue (His-554) was mutated to a glutamine to disable its phosphoryl transfer activity. The new construct was verified by DNA sequencing and then subcloned into a modified pET-32a vector (14) to form a thioredoxin fusion protein with a His 6 tag. After transformation with an expression vector, Escherichia coli strain BL21(DE3) (Novagen) was grown in either Luria Bertini or minimal media (with 15 NH 4 Cl and/or 13 C 6 -glucose as the sole nitrogen or carbon source, respectively), induced with 1 mM isopropyl--D-thiogalactopyranoside at an A 600 of ϳ0.8, and harvested by centrifugation after 4 h of induction. After harvesting, the cell pellet was resuspended in 50 ml (per liter of culture) of 50 mM Tris, pH 7.4, 100 mM NaCl, 2 mM -mercaptoethanol, 10 mM imidazole, and 1 mM phenylmethylsulfonyl fluoride. The suspension was lysed by three passages through a microfluidizer and centrifuged at 10,000 ϫ g for 20 min. The supernatant fraction was loaded * This work was supported by the intramural program of NIDDK, National Institutes of Health, and the Intramural AIDS Targeted Antiviral Program of the Office of the Director of the National Institutes of Health (to G. M. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.