The phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) [1,2] is a major carbohydrate transport system in bacteria. The PTS catalyzes the phosphorylation of incoming sugar substrates concomitant with their translocation across the cell membrane. The general mechanism of the PTS is the following: a phosphoryl group from phosphoenolpyruvate (PEP) is transferred to enzyme I (EI) of PTS which in turn transfers it to a phosphoryl carrier protein (HPr) (see <PDOC00318>). Phospho-HPr then transfers the phosphoryl group to a sugar-specific permease which consists of at least three structurally distinct domains (IIA, IIB, and IIC), [3] which can either be fused together in a single polypeptide chain or exist as two or three interactive chains, formerly called enzymes II (EII) and III (EIII).

The first domain (IIA) (see <PDOC00528>), carries the first permease-specific phosphorylation site, an histidine which is phosphorylated by phospho-HPr. The second domain (IIB) is phosphorylated by phospho-IIA on a cysteinyl or histidyl residue, depending on the sugar transported. Finally, the phosphoryl group is transferred from the IIB domain to the sugar substrate concomitantly with the sugar uptake processed by the IIC domain (see <PDOC51103>). The IIC domain forms the translocation channel and the specific substrate-binding site. An additional transmembrane domain IID (see <PDOC51108>), homologous to IIC, can be found in some PTSs, e.g. for mannose [1,3,4,5,6].

According to structural and sequence analyses, the PTS EIIB domain (EC 2.7.1.69) can be divided in five groups [7,8,9,10].

• The PTS EIIB type 1 domain, which is found in the Glucose class of PTS, has an average length of about 80 amino acids. It forms a split α/β sandwich composed of an antiparallel sheet (β 1 to β 4) and three α helices superimposed onto one side of the sheet (see <PDB:1IBA>). The phosphorylation site (Cys) is located at the end of the first β strand on a protrusion formed by the edge of β 1 and the reverse turn between β 1 and β 2 [7].
• The PTS EIIB type 2 domain, which is found in the Mannitol class of PTS, has an average length of about 100 amino acids. It consists of a four stranded parallel β sheet flanked by two α helices (α 1 and 3) on one face and helix α 2 on the opposite face, with a characteristic Rossmann fold comprising two right-handed β-α-β motifs (see <PDB:1VKR>). The phosphorylation site (Cys) is located at the N-terminus of the domain, in the first β strand [8].
• The PTS EIIB type 3 domain, which is found in the Lactose class of PTS, has an average length of about 100 amino acids. It is composed of a central four-stranded parallel open twisted β sheet, which is flanked by three α helices on the concave side and two on the convex side of the β sheet (see <PDB:1IIB>). The phosphorylation site (Cys) is located in the C-terminal end of the first β strand [9].
• The PTS EIIB type 4 domain, which is found in the Mannose class of PTS, has an average length of about 160 amino acids. It has a central core of seven parallel β strands surrounded by a total of six α-helices. Three helices cover the front face, one the back face with the remaining two capping the central β sheet at the top and bottom (see <PDB:1NRZ>). The phosphorylation site (His) is located at the suface exposed loop between strand 1 and helix 1 [10].
• The PTS EIIB type 5 domain, which is found in the Sorbitol class of PTS, has an average length of about 190 amino acids. The phosphorylation site (Cys) is located in the N-terminus of the domain.

The region around the phosphorylated cysteine of some PTS components is well conserved and can be used [11] as a signature pattern for the following IIB domains:

• Arbutin-, cellobiose- and salicin-specific; IIB(Asc).
• β-glucosides-specific; IIB(Bgl).
• Glucose-specific; IIB(Glc).
• N-acetylglucosamine-specific; IIB(Nag).
• Sucrose-specific; IIB(Scr).
• Maltose and glucose-specific (gene malX).
• Trehalose-specific (gene treB).
• Escherichia coli arbutin-like (gene glvB).
• Bacillus subtilis sacX.

A EIIB-like type 2 domain can be found in bacterial transcriptional regulatory proteins [5]. In these cases, the EIIB-like domain is found in association with other domains like the DeoR-type HTH domain (see <PDOC00696>) or the PTS regulatory domain (a transcriptional antiterminator). It may possess a regulatory function, through its phosphorylation activity, or act as a simple phosphoryl donor.

We have developed a signature pattern for the phosphorylation site of EIIB domains. We also developed five profiles that cover the entire PTS EIIB domains. These profiles are directed respectively against Glucose class of PTS, Mannitol class of PTS, Lactose class of PTS, Mannose class of PTS, and Sorbitol class of PTS.