THE STRUCTURE OF ENZYME IIA(LACTOSE) FROM LACTOCOCCUS-LACTIS REVEALS A NEW FOLD AND POINTS TO POSSIBLE INTERACTIONS OF A MULTICOMPONENT SYSTEM

Background: The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) is responsible for the binding, transmembrane transport and phosphorylation of numerous sugar substrates. The system is also i nvolved in the regulation of a variety of metabolic and transcriptiona l processes. The PTS consists of two non-specific energy coupling comp onents, enzyme I and a heat stable phosphocarrier protein (HPr), as we ll as several sugar-specific multiprotein permeases known as enzymes I I. In most cases, enzymes IIA and IIB are located in the cytoplasm, wh ile enzyme IIC acts as a membrane channel. Enzyme IIA(lactose) belongs to the lactose/cellobiose-specific family of enzymes II, one of four functionally and structurally distinct groups, The protein, which norm ally functions as a trimer, is believed to separate into its subunits after phosphorylation. Results: The crystal structure of the trimeric enzyme IIA(lactose) from Lactococcus lactis has been determined at 2.3 Angstrom resolution. The subunits of the enzyme, related to each othe r by the inherent threefold rotational symmetry, possess interesting s tructural features such as coiled-coil-like packing and a methionine c luster. The subunits each comprise three helices (I, II and III) and p ack against each other forming a nine-helix bundle, This helical bundl e is stabilized by a centrally located metal ion and also encloses a h ydrophobic cavity, The three phosphorylation sites (His78 on each mono mer) are located in helices III and their sidechains protrude into a l arge groove between helices I and II of the neighbouring subunits, A m odel of the complex between phosphorylated HPr and enzyme IIA(lactose) has been constructed. Conclusions: Enzyme IIA(lactose) is the first r epresentative of the family of lactose/cellobiose-specific enzymes IIA for which a three-dimensional structure has been determined. Some of its structural features, like the presence of two histidine residues a t the active site, seem to be common to all enzymes IIA, but no overal l structural homology is observed to any PTS proteins or to any other proteins in the Protein Data Bank, Enzyme IIA(lactose) shows surface c omplementarity to the phosphorylated form of HPr and several energetic ally favourable interactions between the two molecules can be predicte d.