Mutational Analysis of Invariant Arginines in the IIABMan Subunit of the Escherichia coliPhosphotransferase System

Abstract

The mannose transporter of bacterial phosphotransferase system mediates uptake of mannose, glucose, and related hexoses by a mechanism that couples translocation with phosphorylation of the substrate. Protein phosphorylation plays an important role in energy transduction, signal transduction and enzyme regulation. Ptype ATPases transport cations across the cell membrane and are transiently phosphorylated on an aspartic acid during turnover (1). Protein kinases and protein phosphatases regulate the activity of enzymes and membrane bound receptors by phosphorylation and dephosphorylation (2, 3). In the phosphoenolpyruvate-dependent phosphotransferase system of bacteria (PTS) 1 active transport and signaling are two functions of a protein phosphorylation cascade comprising four phosphoprotein units. The four components, enzyme I, HPr, IIA, and IIB sequentially transfer phosphoryl groups from phosphoenolpyruvate to carbohydrates that are accumulated across the cell membrane by a mechanism coupling translocation to phosphorylation. Whereas enzyme I and HPr are two energy coupling components, IIA and IIB together with the transmembrane IIC (and sometimes an additional IID) units form the sugar-specific transport complexes. IIA, IIB, and IIC occur either as protein subunits or as domains of a multidomain protein. Escherichia coli for example has over 30 genes for transporter complexes that differ in substrate specificity, amino acid sequence, and subunit/domain composition. The phosphorylation sites are histidines in enzyme I, HPr and the different IIA components, cysteines in the IIBs belonging to the glucose, mannitol, and lactose family, and histidines in the IIBs belonging to the mannose family of PTS transporters (for reviews see Refs. 4 and 5).Phosphate-binding sites and phosphate-catalytic sites frequently contain arginines, which by their guanidino group can stabilize phosphate through hydrogen bonding and electrostatic interactions (6). Amino acid sequence comparisons with respect to invariant arginines in PTS proteins from different bacteria reveal the following picture. The N-terminal domain of enzyme I has , which are completely invariant in 17 sequences. are close to the phosphorylation site His-189 (7). Arg-131 is conserved in 14 and Arg-136 is conserved in 15 sequences. The C-terminal domain has 7 completely invariant arginines (Protein Domain Data Base: http://protein.toulouse.inra.fr/prodom/ prodom.html). The functional role of these residues in enzyme I has not been explored. HPr is a 9-kDa open faced fourstranded antiparallel ␤-sheet with three ␣-helices on one face. Arg-17, which is close to the active site His-15, is invariant in all 22 known sequences. When Arg-17 is replaced by Ser or Glu, phospho-donor and -acceptor activity of HPr are reduced to between 6% and less than 0.1% of the control (8). The phosphoryl group bound to His-15 of HPr is most likely complexed by the guanidino group of Arg-17, as inferred from its restricted conformational freedom observed upon phosphorylation of HPr, and fr...

Structure, dynamics and biophysics of the cytoplasmic protein–protein complexes of the bacterial phosphoenolpyruvate: sugar phosphotransferase system

“…This is supported by mutation of Arg172 to Gln which results in a substantial increase in the population of the productive complex; the non-productive complex, however, is not completely eliminated as the mutation still leaves an unfavorable polar residue in the middle of the interface. These observations are consistent with the finding that IIB Man ( R172Q ) is less efficiently phosphorylated than the wild type by IIA Man [82]. …”

Signature-Tagged Mutagenesis of Klebsiella pneumoniae To Identify Genes That Influence Biofilm Formation on Extracellular Matrix Material

“…These mutations did not affect the ability of the bacteria to grow in and utilize glucose, sucrose, or cellobiose. However, other bacterial phosphotransferase systems exist within Klebsiella for the transport of glucose, sucrose, and cellobiose (25,31,58). Future work will be aimed at determining if the mutations in these genes affect transport of other specific sugars.…”

The gene encoding IIABL Man in Streptococcus salivarius is part of a tetracistronic operon encoding a phosphoenolpyruvate:mannose/glucose phosphotransferase system The GenBank accession number for the sequence reported in this paper is AF130465.

“…The five arginine residues reported to be involved in the mannose-PTS activity of the E. coli IIB Man (R "') , R "(# , R "*! , R #!% and R $!% ) (Gutknecht et al, 1998) are strictly conserved, with the exception of IIB Aga and IIB Aga h . Other workers have proposed a signature sequence for the IIB domain of the mannose-PTS family (Reizer et al, 1996) (27 %) and E. coli agaW (23 %).…”