Substitutions at the Asp-473 Latch Residue of Chlamydomonas Ribulosebisphosphate Carboxylase/Oxygenase Cause Decreases in Carboxylation Efficiency and CO2/O2 Specificity

Abstract

The loop between ␣-helix 6 and ␤-strand 6 in the ␣/␤-barrel active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) plays a key role in discriminating between gaseous substrates CO 2 and O 2 . Based on numerous x-ray crystal structures, loop 6 is either closed or open depending on the presence or absence, respectively, of substrate ligands. The carboxyl terminus folds over loop 6 in the closed conformation, prompting speculation that it may trigger or latch loop 6 closure. Because an x-ray crystal structure of tobacco Rubisco revealed that phosphate is located at a site in the open form that is occupied by the carboxyl group of Asp-473 in the closed form, it was proposed that Asp-473 may serve as the latch that holds the carboxyl terminus over loop 6. To assess the essentiality of Asp-473 in catalysis, we used directed mutagenesis and chloroplast transformation of the green alga Chlamydomonas reinhardtii to create D473A and D473E mutant enzymes. The D473A and D473E mutant strains can grow photoautotrophically, indicating that Asp-473 is not essential for catalysis. However, both substitutions caused 87% decreases in carboxylation catalytic efficiency (V max /K m ) and ϳ16% decreases in CO 2 /O 2 specificity. If the carboxyl terminus is required for stabilizing loop 6 in the closed conformation, there must be additional residues at the carboxyl terminus/loop 6 interface that contribute to this mechanism. Considering that substitutions at residue 473 can influence CO 2 /O 2 specificity, further study of interactions between loop 6 and the carboxyl terminus may provide clues for engineering an improved Rubisco.Like many ␣/␤-barrel-domain enzymes (1), the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, 1 EC 4.1.1.30) has a loop (i.e. between ␤-strand 6 and ␣-helix 6) that folds over substrate during catalysis (reviewed in Refs. 2-4). Numerous studies have indicated that loop 6 plays a major role in discriminating between CO 2 and O 2 in the competing RuBP carboxylation and oxygenation reactions of Rubisco (reviewed in Refs. 2 and 3), and Lys-334 at the apex of loop 6 interacts with the C-2 carboxyl group of the transition state analog CABP in various Rubisco x-ray crystal structures (5-9). However, Rubisco may be unique among ␣/␤-barrel enzymes in that the carboxyl terminus folds over loop 6 and appears to stabilize its closed conformation. This arrangement of loop 6 and the carboxyl terminus is also observed in the crystal structure of unactivated Rubisco that contains RuBP in the active site (10). In vivo, Rubisco activase is responsible for facilitating the opening of the closed structure to release this RuBP (reviewed in Refs. 2 and 11), thereby allowing spontaneous carbamylation of an active site Lys-201 and introduction of Mg 2ϩ to produce the active form of the enzyme (reviewed in Ref. 12). The change from closed to open conformation in the carboxyl-terminal, ␣/␤-barrel domain of the large subunit is accompanied by movement of the amino-terminal domain ...

Structure-Function Studies with the Unique Hexameric Form II Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) from Rhodopseudomonas palustris

“…5). The active sites of sulfate-bound and CABP-bound R. palustris form II Rubisco structures are virtually superimposable with the exception of the position of residue Glu 49 , which appears to be closer to all other active site residues in the sulfate-bound structure (Fig. 5A).…”

“…In form I Rubisco, the loop 6/carboxyl terminus arrangement has been linked to the regulation of active site opening and closure and is thus believed to be critical for optimal rates of catalysis (53). However, disruption of these interactions via substitutions at a conserved carboxyl-terminal residue could still be tolerated for the function of the enzyme in vivo in Chlamydomonas (49). When the active site is unliganded or occupied by loosely bound substrates, the carboxyl terminus is either disordered or pointed away from loop 6 (3, 7).…”

“…Several studies have established the carboxyl terminus as a hot spot for affecting Rubisco catalysis (47)(48)(49). Structure comparisons and unique placement of the carboxyl terminus in the activated CABP-bound R. palustris Rubisco (Fig.…”

“…Substitution with a bulkier Val would be expected to push ␣B (with A47V) toward the active site. Glu 49 of ␣B functions to stabilize Lys 330 and assists in loop 6 closure during catalysis, and Thr 54 in the adjoining loop is involved in binding the P1 phosphate of the substrate (53). Hence, the movement of ␣B likely nudges these catalytically important residues along with Asn 112 closer to the active site (Fig.…”

Functional Hybrid Rubisco Enzymes with Plant Small Subunits and Algal Large Subunits

“…Chlamydomonas has served as a useful genetic model for the study of Rubisco because host strains have been isolated that lack chloroplast rbcL genes and nuclear rbcS genes (35,36) and because Rubisco-deficient mutants can be maintained with acetate as an alternative source of carbon and energy (reviewed in Refs. 1, 5, and 16).…”