CHARACTERIZATION OF A BACTERIAL GENE ENCODING AN AUTOPHOSPHORYLATING PROTEIN-TYROSINE KINASE

Acinetobacter johnsonii harbors a protein tyrosine kinase activity tha t is able to catalyze autophosphorylation, like a number of eukaryotic tyrosine kinases. A biochemical and genetic analysis of this enzyme w as performed. Maximum phosphorylation in vitro was obtained by incubat ing the kinase for 2 min at pH 7.0 in the presence of 5 mM magnesium c hloride. In contrast to eukaryotic enzymes, no inhibitory effect of ge nistein and no phosphorylation of synthetic substrates such as poly (G lu(80) Tyr(20)) or angiotensin II were observed. The analysis of the b acterial kinase by two-dimensional gel electrophoresis revealed the pr esence of at least five isoforms, all phosphorylated exclusively at ty rosine, which supports the concept that autophosphorylation occurs at multiple sites within the protein. The cloning and nucleotide sequenci ng of the gene encoding this kinase were achieved, which represents th e first molecular characterization of a gene of this type in bacteria. An open reading frame of 2199 nucleotides encoding a protein of 82 37 3 Da was detected. The analysis of the deduced amino acid sequence sug gested a possible involvement of the enzyme in cell recognition and ba cterial pathogenicity. In addition, the cloning and sequencing of the region immediately upstream of the gene encoding the kinase revealed a novel open reading frame of 426 nucleotides encoding a phosphotyrosin e protein phosphatase of 16 217 Da, which indicates that autophosphory lation on tyrosine is a physiologically reversible reaction. (C) 1997 Elsevier Science B.V.