Mutations of an active site threonyl residue promote beta elimination and other side reactions of the enediol intermediate of the ribulosebisphosphate carboxylase reaction.

Morell, Matthew K.; Paul, Kalanethee; O'Shea, N J; Kane, Heather J.; Andrews, T. John

doi:10.1016/s0021-9258(17)37164-8

Cited by 39 publications

(18 citation statements)

References 42 publications

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“…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).…”

Section: Resultsmentioning

confidence: 95%

“…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 ).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2019

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The enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its central role in capturing atmospheric CO 2 via the Calvin−Benson−Bassham (CBB) cycle have been well-studied. Previously, a form II RuBisCO from Rhodopseudomonas palustris, a facultative anaerobic bacterium, was shown to assemble into a hexameric holoenzyme. Unlike previous studies with form II RuBisCO, the R. palustris enzyme could be crystallized in the presence of the transition state analogue 2-carboxyarabinitol 1,5bisphosphate (CABP), greatly facilitating the structure−function studies reported here. Structural analysis of mutant enzymes with substitutions in form II-specific residues (Ile165 and Met331) and other conserved and semiconserved residues near the enzyme's active site identified subtle structural interactions that may account for functional differences between divergent RuBisCO enzymes. In addition, using a distantly related aerobic bacterial host, further selection of a suppressor mutant enzyme that overcomes negative enzymatic functions was accomplished. Structure−function analyses with negative and suppressor mutant RuBisCOs highlighted the importance of interactions involving different parts of the enzyme's quaternary structure that influenced partial reactions that constitute RuBisCO's carboxylation mechanism. In particular, structural perturbations in an intersubunit interface appear to affect CO 2 addition but not the previous step in the enzymatic mechanism, i.e., the enolization of substrate ribulose 1,5-bisphosphate (RuBP). This was further substantiated by the ability of a subset of carboxylation negative mutants to support a previously described sulfur-salvage function, one that appears to rely solely on the enzyme's ability to catalyze the enolization of a substrate analogous to RuBP.

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Section: Resultsmentioning

confidence: 95%

Section: Resultsmentioning

confidence: 99%

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

et al. 2019

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“…XuBP formation by H287N therefore indicates an inability of the mutant to completely sequester enediol from solvent. Interestingly, DiMP, the other characterized enediolderived side product that has been observed with Rubisco mutants (Harpel & Hamnan, 1994;Larimer et al, 1994;Morel1 et al, 1994;Larson et al, 1995), is absent in H287N reaction profiles (expected elution time -21 min). This dicarbonyl results from &elimination of phosphate from the enediol, either in solution due to its dissociation from the active site, or at the active site due to departure of the intermediate from an extended conformation that maintains the bridge oxygen of the C1-phosphate group coplanar with the double bond (Rose, 1981).…”

Section: Product Analysismentioning

confidence: 95%

Multiple catalytic roles of His 287 of Rhodospirillum rubrum ribulose 1,5‐bisphosphate carboxylase/oxygenase

1998

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Active-site His 287 of Rhodospirillum rubrum ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase interacts with the C3-hydroxyl of bound substrate or reaction-intermediate analogue (CABP), water molecules, and ligands for the activator metal-ion (Anderson I, 1996, J Mol Biol259:160-174 Rubisco links photosynthetic energy capture to the net fixation of atmospheric C 0 2 by catalyzing the carboxylation of RuBP to form two molecules of the 3-carbon carbohydrate precursor, PGA. The overall carboxylation reaction entails a complex scheme of sequential reactions, including enolization of RuBP, addition of C 0 2 and H 2 0 to the 2,3-enediol of RuBP,' C-C cleavage, and stereospecific protonation of one cleavage product (reviewed in Andrews & Schloss, 1990; RuBP, o-ribulose 1,5-bisphosphate; PGA, 3-phospho-o-glycerate; CABP, 2-carboxy-o-arabinitol 1.5-bisphosphate; CKABP, 2-carboxy-3-ketoarabinitol 1,5-bisphosphate; bicine, N, N-bis(2-hydroxyethy1)glycine; PGyc, 2-phosphoglycolate; CTBP, 2-carboxytetritol 1,4-bisphosphate; DiBP, 2,3-pentodiulose 1,5-bisphosphate; XuBP, xylulose 1,5-bisphosphate; DiMP, 1-deoxy-D-glycero 2,3-pentodiulose 5-phosphate.'The deprotonated form of RuBP varies in ionic state between enediol and enediolate forms throughout the reaction coordinate; we will collectively refer to this intermediate as "enediol." carboxylation reaction have appeared recently (Newman & Gutteridge, 1993; Anderson, 1996;Taylor & Anderson, 1997a). Although these mechanisms embrace a wealth of accumulated chemical, kinetic, mutagenic, and structural knowledge about this enzyme, several features remain unresolved, including elucidation of C02/02 specificity determinants and consensus assignment of functionally relevant residues to precise steps of the overall reaction.Among the strictly conserved active-site residues in direct contact with bound substrate or intermediate analogues (Knight et al., 1990;Lundqvist & Schneider, 1991;Newman & Gutteridge, 1993;Schreuder et al., 1993; Anderson, 1996;Taylor & Anderson, 1997a, 1997b, His 2872 has not been subjected to sufficient functional scrutiny to allow its catalytic roles to be defined. Thus, several different mechanistic interpretations of structural data have been offered concerning the function of His 287. This residue approximates the activator site (comprised of a divalent metal-ion coordinated by Asp 193, Glu 194, and the carbamate of Lys 191), contacts bound ligands, and forms hydrogen bonds with active-site 'Residue numbers refer to the sequence position in R. rubrum Rubisco.In the designations for the mutants, the first letter refers to the amino acid found in the wild-type enzyme and the second letter denotes the amino acid replacement. 730

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“…H. neapolitanus Form IA C (HnLS) Rubisco kinetics were distinct, being faster and possessing a much higher Michaelis constant for CO2. Compared to the fast model J o u r n a l P r e -p r o o f carboxysomal Form IB Synechococcus enzyme SynLS (54)(55)(56), HnLS displayed an intermediate phenotype (Table 1).…”

Section: The Inhibition and Reaction Kinetics Of Cbbqo-associated Form Ia Rubiscos Are Diversementioning

confidence: 99%

The CbbQO-type rubisco activases encoded in carboxysome gene clusters can activate carboxysomal form IA rubiscos

Tsai¹,

Liew²,

Guo

et al. 2022

Journal of Biological Chemistry

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Mutations of an active site threonyl residue promote beta elimination and other side reactions of the enediol intermediate of the ribulosebisphosphate carboxylase reaction.

Cited by 39 publications

References 42 publications

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

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

Multiple catalytic roles of His 287 of Rhodospirillum rubrum ribulose 1,5‐bisphosphate carboxylase/oxygenase

The CbbQO-type rubisco activases encoded in carboxysome gene clusters can activate carboxysomal form IA rubiscos

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Mutations of an active site threonyl residue promote beta elimination and other side reactions of the enediol intermediate of the ribulosebisphosphate carboxylase reaction.

Cited by 39 publications

References 42 publications

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

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

Multiple catalytic roles of His 287 of Rhodospirillum rubrum ribulose 1,5‐bisphosphate carboxylase/oxygenase

The CbbQO-type rubisco activases encoded in carboxysome gene clusters can activate carboxysomal form IA rubiscos

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

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