S. Elsen et al., PURIFICATION AND IN-VITRO PHOSPHORYLATION OF HUPT, A REGULATORY PROTEIN CONTROLLING HYDROGENASE GENE-EXPRESSION IN RHODOBACTER-CAPSULATUS, Journal of bacteriology, 179(3), 1997, pp. 968-971
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.