Thomas S. Soper

Overproduction of Anabaena 7120 ribulose-bisphosphate carboxylase/oxygenase in Escherichia coli

As a prerequisite to protein engineering, we have overexpressed the rbcLS operon of the cyanobacterium Anabaena 7120, in Escherichia coli. The operon encodes the large and small subunits of ribulose-bisphosphate carboxylase/oxygenase (Rubisco). Levels of active enzyme exceed 6% of soluble protein. We noted an apparent third gene, an unidentified open reading frame (URF) referred to here as rbcX, in the 558-bp intergenic space between the large and small subunit encoding genes. The URF, rbcX, has no known function. High-level production of Rubisco activity from the rbc operon in E. coli required simultaneous overproduction of the GroESL chaperonins under a regimen of limited growth, in contrast to more modest conditions which suffice for efficient production of the Anacystis nidulans cyanobacterial Rubisco. Deletion of rbcX or inversion of the rbcL-rbcS order did not enhance expression levels. The recombinant Rubisco, purified to near homogeneity, exhibited functional properties [Km(ribulose-P2), kcat, transition-state-analogue binding stoichiometry/exchange, and specificity factor] essentially identical to those of the enzyme obtained from Anabaena.

Essentiality of Glu-48 of ribulose bisphosphate carboxylase/oxygenase as demonstrated by site-directed mutagenesis.

Previous reports provide indirect evidence for the presence of Glu-48 at the active site of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. This possibility has been examined directly by replacement of Glu-48 with glutamine via site-directed mutagenesis. This single amino acid substitution does not prevent subunit association or ligand binding. However, the Glu-48 mutant is severely deficient in catalytic activity, exhibiting a kcat only 0.05% that of wild-type enzyme. These results demonstrate that Glu-48 is positioned at the active site and suggest that it serves a functional role. In conjunction with previous studies, the discovery of essentiality of Glu-48 argues that the active site is located at an interface between subunits.

Examination of subunit interactions at the active site of ribulose 1,5-bisphosphate carboxylase/oxygenase fromRhodospirillum rubrum by hybridization of site-directed mutants

The two active sites of homodimeric ribulose bisphosphate carboxylase/oxygenase fromRhodospirillum rubrum are constituted by interacting domains of adjacent subunits, in which residues from each are required for catalytic activity. Active-site residues include Lys-166 of one domain and Glu-48 of the interacting domain from the adjacent subunit. Whereas all substitutions for Lys-166, introduced by site-directed mutagenesis, abolished catalytic activity, only a negatively charged residue (e.g., aspartic acid) resulted in the disruption of the subunit interactions (Lee et al., 1987). This disruption could result from improper folding of the individual polypeptide chains or to more localized effects (e.g., charge-charge repulsion due to proximal negative charges of Asp-166 and Glu-48 of adjacent domains or conformational changes restricted to a single domain). To address these questions, we have examined the ability of the Asp-166 mutant subunit to associate with a mutant subunit in which the negatively charged Glu-48 has been replaced by the neutral glutaminyl residue. Coexpression inEscherichia coli of the genes for both mutant subunits results in formation of a catalytically active hybrid, despite the absence of activity when either gene is expressed individually. Isolation and characterization of the hybrid show that it is composed of one Asp-166 subunit and one Gln-48 subunit, presumably with only one functional active site per dimeric molecule. This association of dissimilar subunits shows that introduction of a negative charge at position 166 does not lead to overall distortion of subunit conformation. In contrast to the wild-type enzyme, the hybrid dissociates spontaneously at low protein concentration but is stablized by elevated ionic strengths or by glycerol.

Site-directed mutagenesis to determine essential residues of ribulose- bisphosphate carboxylase of Rhodospirillum rubrum

Both Lys-166 and His-291 of ribulosebisphosphate carboxylase/oxygenase fromRhodospirillum rubrum have been implicated as the active-site residue that initiates catalysis. To decide between these two candidates, we resorted to site-directed mutagenesis to replace Lys-166 and His-291 with several amino acids. All 7 of the position-166 mutants tested are severely deficient in carboxylase activity, whereas the alanine and serine mutants at position 291 are ∼40% and ∼18% as active as the native carboxylase, essentially ruling out His-291 in theRhodospirillum rubrum carboxylase (and by inference His-298 in the spinach enzyme) as a catalytically essential residue. The ability of some of the mutant proteins to undergo carbamate formation or to bind either ribulosebisphosphate or a transition-state analogue remains largely unimpaired. This implies that Lys-166 is not required for substrate binding; rather, the results corroborate the earlier postulate that Lys-166 functions as an acid-base group in catalysis or in stabilizing a transition state in the reaction pathway.