Involvement of a protein tyrosine kinase in production of the polymeric bioemulsifier emulsan from the oil-degrading strain Acinetobacter lwoffii RAG-1.
The genes associated with the biosynthesis of the polymeric bioemulsifier emulsan, produced by the oil-degrading Acinetobacter lwoffii RAG-1 are clustered within a 27-kbp region termed the wee cluster. This report demonstrates the involvement of two genes of the wee cluster of RAG-1, wzb and wzc, in emulsan biosynthesis. The two gene products, Wzc and Wzb were overexpressed and purified. Wzc exhibited ATP-dependent autophosphorylating protein tyrosine kinase activity. Wzb was found to be a protein tyrosine phosphatase capable of dephosphorylating the phosphorylated Wzc. Using the synthetic substrate p-nitrophenyl phosphate (PNPP) Wzb exhibited a V(max) of 12 micromol of PNPP min(-1) mg(-1) and a K(m) of 8 mM PNPP at 30 degrees C. The emulsifying activity of mutants lacking either wzb or wzc was 16 and 15% of RAG-1 activity, respectively, suggesting a role for the two enzymes in emulsan production. Phosphorylation of Wzc was found to occur within a cluster of five tyrosine residues at the C terminus. Colonies from a mutant in which these five tyrosine residues were replaced by five phenylalanine residues along with those of a second mutant, which also lacked Wzb, exhibited a highly viscous colony consistency. Emulsan activity of these mutants was 25 and 24% of that of RAG-1, respectively. Neither of these mutants contained cell-associated emulsan. However, they did produce an extracellular high-molecular-mass galactosamine-containing polysaccharide. A model is proposed in which subunit polymerization, translocation and release of emulsan are all associated and coregulated by tyrosine phosphorylation.
Nakar
David
D
Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Science, Tel Aviv University, Tel Aviv 69978, Israel.
Gutnick
David L
DL
D000150:Acinetobacter / Q000378:metabolism*; D001426:Bacterial Proteins / Q000502:physiology*; D008565:Membrane Proteins; D010766:Phosphorylation; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D017027:Protein Tyrosine Phosphatases / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000502:physiology*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D011135:Polysaccharides, Bacterial; C417168:WZC protein, Bacteria; C036028:emulsan; D011505:Protein-Tyrosine Kinases; D017027:Protein Tyrosine Phosphatases