Molecular Approaches to Probe Differential NADH Activation of Phosphoribulokinase Isozymes fromRhodobacter sphaeroides

Abstract

ThecbbPIandcbbPIIgenes fromRhodobacter sphaeroides,encoding highly similar phosphoribulokinase (PRK) isozymes, PRK I and PRK II, respectively, exhibited differential allosteric activation by NADH. The twocbbPgenes were cloned into expression vectors and homogeneous recombinant protein prepared. PRK II was found to be inherently less stable than PRK I; however, the addition of substrate ATP resulted in the complete protection of both isozymes to a 15-min incubation at 50°C. The relative molecular masses for both octameric isozymes were determined to be approximately 230,000; however, the protective effect of ATP was in accordance with aggregation of monomers to a molecular mass of approximately 750,000. While PRK I exhibited a nearly absolute dependence upon NADH for activity, PRK II retained substantial activity in the absence of NADH. PRK chimeras were thus constructed to facilitate elucidation of the basis for the differential effect of NADH, with advantage taken of the relative sequence identity of about 90% between the two isozymes. Chimeras were constructed either byin vivohomologous recombination, using thesacBgene fromBacillus subtilisas a conditionally lethal marker, or by using convenient restriction sites to combine different parts of the twocbbPgenes. The PRK chimeras generated contained either the amino-terminal domain of PRK II and the carboxy-terminal domain of PRK I or the opposite configuration. Subsequent analyses of the chimeras pointed to particular regions and residue(s) as likely being important for NADH activation.