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Plant-like substitutions in the large-subunit carboxy terminus of Chlamydomonas Rubisco increase CO2/O2 specificity.

Satagopan S, Spreitzer RJ - BMC Plant Biol. (2008)

Bottom Line: The mutations do not seem to influence the protein expression, structural stability or the function in vivo.Owing to the decreased carboxylation catalytic efficiency, the quadruple-mutant is not a "better" enzyme.Nonetheless, because of its positive influence on specificity, the carboxy terminus, relatively far from the active site, may serve as a target for enzyme improvement via combinatorial approaches.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Ohio State University, Columbus, OH 43210, USA. satagopan.1@osu.edu

ABSTRACT

Background: Ribulose-1,5-bisphosphate is the rate-limiting enzyme in photosynthesis. The catalytic large subunit of the green-algal enzyme from Chlamydomonas reinhardtii is approxiamtely 90% identical to the flowering-plant sequences, although they confer diverse kinetic properties. To identify the regions that may account for species variation in kinetic properties, directed mutagenesis and chloroplast transformation were used to create four amino-acid substitutions in the carboxy terminus of the Chlamydomonas large subunit to mimic the sequence of higher-specificity plant enzymes.

Results: The quadruple-mutant enzyme has a 10% increase in CO2/O2 specificity and a lower carboxylation catalytic efficiency. The mutations do not seem to influence the protein expression, structural stability or the function in vivo.

Conclusion: Owing to the decreased carboxylation catalytic efficiency, the quadruple-mutant is not a "better" enzyme. Nonetheless, because of its positive influence on specificity, the carboxy terminus, relatively far from the active site, may serve as a target for enzyme improvement via combinatorial approaches.

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Related in: MedlinePlus

Comparison of structural interactions at the carboxy-terminal/loop-6 interface in the large subunit of Rubisco from (A) Chlamydomonas (1GK8) [9] and (B) spinach (8RUC) [10]. Residues within 4 Å of the divergent carboxy-terminal residues 470, 471, 472, and 474 are shown as sticks. The carboxy terminus (yellow), loop 6 (red), and part of a loop in the amino-terminal domain of a neighboring large subunit (blue) are drawn as ribbons. Residues not in these three structural regions are colored green. The location of the active site is denoted by Lys-334 and the transition-state analog CABP. Potential hydrogen and ionic bonds are indicated by red and black dotted lines, respectively, connecting the participating atoms.
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Figure 1: Comparison of structural interactions at the carboxy-terminal/loop-6 interface in the large subunit of Rubisco from (A) Chlamydomonas (1GK8) [9] and (B) spinach (8RUC) [10]. Residues within 4 Å of the divergent carboxy-terminal residues 470, 471, 472, and 474 are shown as sticks. The carboxy terminus (yellow), loop 6 (red), and part of a loop in the amino-terminal domain of a neighboring large subunit (blue) are drawn as ribbons. Residues not in these three structural regions are colored green. The location of the active site is denoted by Lys-334 and the transition-state analog CABP. Potential hydrogen and ionic bonds are indicated by red and black dotted lines, respectively, connecting the participating atoms.

Mentions: The loop between β-strand 6 and α-helix 6 of the large-subunit α/β barrel folds over the transition-state analog carboxyarabinitol 1,5-bisphosphate (CABP) [8] (Fig. 1). In this closed conformation, the large-subunit carboxy terminus (residues Trp-462 to Leu-475) packs over loop 6 (Fig. 1) [9,10]. In Chlamydomonas Rubisco, substitution of conserved Asp-473, which was proposed to be an essential latch residue [11], with either Ala or Glu did not eliminate catalysis but caused a 14–17% decrease in Ω [12]. Deletion of 10 residues from the carboxy terminus of the Synechococcus (cyanobacterium) large subunit caused a 38% decrease in Ω, and a group of four plant-like substitutions E470P/T471A/K474T/L475V was reported to cause a 9% increase in Ω [13]. However, this small increase was close to the experimental error of the assays employed [13]. Lengthening the carboxy terminus beyond residue 475 had no effect on Ω [14].


Plant-like substitutions in the large-subunit carboxy terminus of Chlamydomonas Rubisco increase CO2/O2 specificity.

Satagopan S, Spreitzer RJ - BMC Plant Biol. (2008)

Comparison of structural interactions at the carboxy-terminal/loop-6 interface in the large subunit of Rubisco from (A) Chlamydomonas (1GK8) [9] and (B) spinach (8RUC) [10]. Residues within 4 Å of the divergent carboxy-terminal residues 470, 471, 472, and 474 are shown as sticks. The carboxy terminus (yellow), loop 6 (red), and part of a loop in the amino-terminal domain of a neighboring large subunit (blue) are drawn as ribbons. Residues not in these three structural regions are colored green. The location of the active site is denoted by Lys-334 and the transition-state analog CABP. Potential hydrogen and ionic bonds are indicated by red and black dotted lines, respectively, connecting the participating atoms.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2527014&req=5

Figure 1: Comparison of structural interactions at the carboxy-terminal/loop-6 interface in the large subunit of Rubisco from (A) Chlamydomonas (1GK8) [9] and (B) spinach (8RUC) [10]. Residues within 4 Å of the divergent carboxy-terminal residues 470, 471, 472, and 474 are shown as sticks. The carboxy terminus (yellow), loop 6 (red), and part of a loop in the amino-terminal domain of a neighboring large subunit (blue) are drawn as ribbons. Residues not in these three structural regions are colored green. The location of the active site is denoted by Lys-334 and the transition-state analog CABP. Potential hydrogen and ionic bonds are indicated by red and black dotted lines, respectively, connecting the participating atoms.
Mentions: The loop between β-strand 6 and α-helix 6 of the large-subunit α/β barrel folds over the transition-state analog carboxyarabinitol 1,5-bisphosphate (CABP) [8] (Fig. 1). In this closed conformation, the large-subunit carboxy terminus (residues Trp-462 to Leu-475) packs over loop 6 (Fig. 1) [9,10]. In Chlamydomonas Rubisco, substitution of conserved Asp-473, which was proposed to be an essential latch residue [11], with either Ala or Glu did not eliminate catalysis but caused a 14–17% decrease in Ω [12]. Deletion of 10 residues from the carboxy terminus of the Synechococcus (cyanobacterium) large subunit caused a 38% decrease in Ω, and a group of four plant-like substitutions E470P/T471A/K474T/L475V was reported to cause a 9% increase in Ω [13]. However, this small increase was close to the experimental error of the assays employed [13]. Lengthening the carboxy terminus beyond residue 475 had no effect on Ω [14].

Bottom Line: The mutations do not seem to influence the protein expression, structural stability or the function in vivo.Owing to the decreased carboxylation catalytic efficiency, the quadruple-mutant is not a "better" enzyme.Nonetheless, because of its positive influence on specificity, the carboxy terminus, relatively far from the active site, may serve as a target for enzyme improvement via combinatorial approaches.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Ohio State University, Columbus, OH 43210, USA. satagopan.1@osu.edu

ABSTRACT

Background: Ribulose-1,5-bisphosphate is the rate-limiting enzyme in photosynthesis. The catalytic large subunit of the green-algal enzyme from Chlamydomonas reinhardtii is approxiamtely 90% identical to the flowering-plant sequences, although they confer diverse kinetic properties. To identify the regions that may account for species variation in kinetic properties, directed mutagenesis and chloroplast transformation were used to create four amino-acid substitutions in the carboxy terminus of the Chlamydomonas large subunit to mimic the sequence of higher-specificity plant enzymes.

Results: The quadruple-mutant enzyme has a 10% increase in CO2/O2 specificity and a lower carboxylation catalytic efficiency. The mutations do not seem to influence the protein expression, structural stability or the function in vivo.

Conclusion: Owing to the decreased carboxylation catalytic efficiency, the quadruple-mutant is not a "better" enzyme. Nonetheless, because of its positive influence on specificity, the carboxy terminus, relatively far from the active site, may serve as a target for enzyme improvement via combinatorial approaches.

Show MeSH
Related in: MedlinePlus