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 alpha helix region of the eight-stranded beta/alpha barrel domain of the large subunit of Synechococcus sp. strain PCC 6301 ribulosebisphosphate carboxylase/oxygenase (rubisco) was altered by site-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 (ratio of the rate constants for the carboxylase and oxygenase reactions). A set of cumulative mutations of one to eight amino acid residues was prepared and examined and it was found that mutant enzymes which contained from one to five substitutions all exhibited substantial decreases 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 their native structure, consecutive changes in the alpha helix 6 region yielded a progressive increase in the K(m) for ribulosebisphosphate, confirming the importance of this region in substrate binding. Despite these results, and previous studies which indicated the importance and potential of residues in the alpha helix 6 region to influence the ability of loop 6 to affect rubisco catalysis, simple cumulative substitution did not significantly alter the substrate specificity factor of the enzyme. The results of this study lend further credence to the idea that engineered enhancement of rubisco specificity will likely require coordination of alterations at multiple sites in the primary structure.
Ramage
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Department of Microbiology and Plant Molecular Biology/Biotechnology Program, Ohio State University, Columbus 43210-1292, USA.
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D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D000458:Cyanobacteria / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D012273:Ribulose-Bisphosphate Carboxylase