Cloning and characterization of the pknA gene from Streptomyces coelicolorA3(2), coding for the Mn2+-dependent protein Ser/Thr kinase

A gene pknA, coding for an eukaryotic-type protein Ser/Thr kinase, was clon ed from the Streptomyces coelicolor A3(2) chromosome. The PknA protein kina se, containing the C-terminal eukaryotic-type kinase domain with an N-termi nal extension, was expressed in Escherichia coli and Streptomyces lividans. The affinity purified MBP-PknA fusion protein was assayed for kinase activ ity that showed its ability to autophosphorylate in vitro in the presence o f [gamma-P-32]ATP. The activity was Mn2+-dependent, The preautophosphorylat ed kinase phosphorylated at least two proteins (sizes 30 and 32 kDa) in the S. coelicolor J1501 cell-fi ee extracts of all developmental stages. The l arger of them was also phosphorylated in vitro by an endogenous protein kin ase in late stages extracts, but not earlier. Although Mn2+-dependent prote in phosphorylation has previously been described In Streptomyces, this is t he first report of a gene encoding such an enzyme in this genus. (C) 2000 A cademic Press.