Site-specific mutagenesis of ribulose-1,5-bisphosphate carboxylase/oxygenase. Evidence that carbamate formation at Lys 191 is required for catalytic activity.

Structural Perturbations of Rhodopseudomonas palustris Form II RuBisCO Mutant Enzymes That Affect CO2 Fixation

“…Steric interactions in this region likely play a role in correctly positioning the carbamylated Lys192 and Ser369 catalytic residues (Lys201 and Ser379 in spinach RuBisCO) (Figure A). Carbamylated Lys192 is essential for magnesium coordination, RuBP proton abstraction, and stabilizing the RuBP 2,3-ene-diol­(ate) intermediate. ,− ,, Ser369 is involved in CO 2 /O 2 specificity and later aspects of the carboxylation mechanism. ,, When Ile165 is substituted with threonine, the side chain hydroxyl moves within hydrogen-bonding distance of O2 of CABP and a carbamyl oxygen of Lys192, much like the arrangement in the CABP-bound spinach enzyme (Figure B,C). Even though this appears to stabilize the position of Lys192, reorientation of Ser369 also results from the loss of a hydrogen bond between the hydroxyl group of Ser369 and O4 of CABP in exchange for a new hydrogen bond with the hydroxyl group of Thr392 (Figure B).…”

“…Conserved catalytic residues Glu49, Thr54, Asn112, Lys167, Lys169, Lys192, and Lys330 (Glu60, Thr65, Asn123, Lys175, Lys177, Lys201, and Lys334 in spinach RuBisCO) have all been implicated in various steps of RuBisCO’s catalytic mechanism on the basis of previous biochemical studies. ,,− To delineate the role of these residues in RuBP enolization versus CO 2 fixation steps of the catalytic mechanism, we constructed mutant enzymes E49A, E49G, T54A, N112A, N112K, N112Q, N112S, K167A, K167G, K167R, K169A, K192A, K192C, K192R, and K330A. The identities of amino acid substitutions introduced were largely prompted by previous structure–function studies with the analogous R. rubrum form II RuBisCO. ,− None of these R. palustris form II RuBisCO mutant enzymes restored CO 2 -dependent growth of R. capsulatus strain SB I/II – (Table and Figure S1), demonstrating compromised RuBP carboxylase function. Upon expression in R. rubrum strain IR, photoheterotrophic growth on MTA remained poor for mutants N112S, K167A, K167G, K192A, K192C, and K192R, identical to that seen for the empty plasmid control ( P > 0.05; df = 4) (Table ).…”

“…Unlike Ile165, which resides in the second sphere of coordination around the active site, residue Lys192 has a direct role in catalysis. Activation of the enzyme via ε-amino carbamylation of this catalytic lysine is necessary for both the RuBisCO carboxylation mechanism and the enolization mechanism of at least one RLP class, i.e., the Bacillus family YkrW/MtnW RLP. − A previous study showed that substitution of the equivalent Lys191 in R. rubrum RuBisCO with cysteine resulted in a mutant enzyme devoid of CO 2 fixation activity but still retained tight binding with CABP, a property characteristic of activated RuBisCOs. , This was the rationale for the structural analysis of R. palustris K192C mutant RuBisCO. The mutant enzyme was indeed amenable to crystallization with CABP, and the structure showed that the absence of an ε-amino group on this residue resulted in the loss of coordination with CABP via the magnesium ion (Figure D).…”

“…Catalytic residue Lys192 (Lys201 in spinach RuBisCO) is responsible for both coordinating the magnesium ion necessary for catalysis and serving as the essential base for C3 proton abstraction of RuBP. ,, Substitutions at this lysine residue render the enzyme devoid of all catalytic activity, including enolization of RuBP and carboxylation of the 2,3-ene-diol­(ate) intermediate, unless a chemical modification mimicking the carbamylated lysine is reintroduced. − The presence of a carbonate ion in the active site of the K192C mutant structure in place of the carbamyl group usually present in the wild-type enzyme (Figure D) points to the requirement of electronegative groups to stabilize the cofactor magnesium ion, which is central to the enolization mechanism. However, rearrangement of multiple interactions around the active site explains the inability of this mutant to participate in key catalytic events that may be required for either function.…”

“…Carbamylated Lys192 is essential for magnesium coordination, RuBP proton abstraction, and stabilizing the RuBP 2,3-ene-diol(ate) intermediate. 6,[55][56][57]61,62 Ser369 is involved in CO 2 /O 2 specificity and later aspects of the carboxylation mechanism. 45,60,61 When Ile165 is substituted with threonine, the side chain hydroxyl moves within hydrogen-bonding distance of O2 of CABP and a carbamyl oxygen of Lys192, much like the arrangement in the CABPbound spinach enzyme (Figure 4B,C).…”