ALTERATION OF THE ALPHA-HELIX REGION OF CYANOBACTERIAL RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE TO REFLECT SEQUENCES FOUND IN HIGH SUBSTRATE-SPECIFICITY ENZYMES/

The sequence at the a helix region of the eight-stranded beta/alpha ba rrel domain of the large subunit of Synechococcus sp. strain PCC 6301 ribulosebisphosphate carboxylase/oxygenase (rubisco) was altered by si te-directed mutagenesis. Changes were made to match the corresponding residues in the rubisco large subunit of chromophytic and rhodophytic algae, which have considerably higher substrate specificity factors (r atio of the rate constants for the carboxylase and oxygenase reactions ). A set of cumulative mutations of one to eight amino acid residues w as prepared and examined and it was found that mutant enzymes which co ntained from one to five substitutions all exhibited substantial decre ases in carboxylase activity. Mutant enzymes which contained from six to eight amino acid substitutions were inactive and failed to maintain their native quarternary structure, For enzymes which maintained thei r native structure, consecutive changes in the alpha helix 6 region yi elded a progressive increase in the K-m for ribulosebisphosphate, conf irming the importance of this region in substrate binding. Despite the se results, and previous studies which indicated the importance and po tential of residues in the alpha helix 6 region to influence the abili ty of loop 6 to affect rubisco catalysis, simple cumulative substituti on did not significantly alter the substrate specificity factor of the enzyme, The results of this study lend further credence to the idea t hat engineered enhancement of rubisco specificity will likely require coordination of alterations at multiple sites in the primary structure . (C) 1998 Academic Press.