PURIFICATION AND IN-VITRO PHOSPHORYLATION OF HUPT, A REGULATORY PROTEIN CONTROLLING HYDROGENASE GENE-EXPRESSION IN RHODOBACTER-CAPSULATUS

The HupT protein of Rhodobacter capsulatus, involved in negative regul ation of hydrogenase gene expression, is predicted to be a histidine k inase on the basis of sequence comparisons. The protein was overproduc ed in Escherichia coil, purified to homogeneity, and demonstrated to a utophosphorylate in vitro in the presence of [gamma-P-32]ATP. An H217N hupt mutant was constructed, and the mutant protein was shown to have lost kinase activity. This result, and the fact that the phosphoryl g roup in phosphorylated HupT appeared to be bound to an N atom, support the suggestion from sequence comparisons that HupT is a histidine kin ase, which can autophosphorylate on the His(217) residue.