Characterization of the Rhizobiaceae protein RhaK
Rivers, Damien M R
MetadataShow full item record
In Rhizobium leguminosarum the ABC transporter responsible for rhamnose transport is dependent on RhaK, a sugar kinase that is necessary for the catabolism of rhamnose. It was hypothesized that RhaK has two separate functions; phosphorylation of rhamnose, and an unknown interaction with the rhamnose ABC transporter. To address this hypothesis a linker-scanning mutagenesis of rhaK was carried out. Two generated variants (RhaK72 and RhaK73) were found to maintain kinase activity, but were severely impaired in rhamnose transport function. Structural modelling suggested that both RhaK72 and RhaK73 affect surface exposed residues in two distinct regions localized to one face of the protein. This suggests that this proteins face may play a role in a protein-protein interaction that affects rhamnose transport. Using a two-hybrid system, an N-terminal and a C-teminal fragment of RhaK were both shown to interact with the N-terminal fragment of RhaT. These fragments span the regions that contain the rhaK73 and rhaK72 inserts respectively. When the rhaK72 and rhaK73 insert alleles were cloned and assayed using the two-hybrid system, these they were unable to interact with the RhaT fragment, suggesting these inserts abolish transport by interfering with a physical interaction between RhaT and RhaK. A phylogeny was generated based on the amino acid sequence of RhaK like proteins found in syntenous opereons. To gain insight into what residues may constitute a binding domain a PRALINE alignment of the orthologous kinases was combined with secondary structure analysis, known informative mutations, and functional residue predictions. A putative 12 amino acid binding site was identified using this method. An alanine scanning mutagenesis and subsequent two-hybrid analysis was carried out on this region. The substitution of any of these residues greatly affected the interaction between RhaT and RhaK. Although heterologous complementation of RhaK is possible, cosmid complementation anomalies and phylogenetic analysis of RhaK indicates the R. leguminosarum and S. meliloti kinases are different. Through a series of heterologous complementation experiments, enzyme assays, gene fusions, and transport experiments we show that the R. leguminosarum kinase is capable of directly phosphorylating rhamnose and rhamnulose, whereas the Sinorhizobium meliloti kinase does not have rhamnose kinase activity.