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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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Carboxy-terminal substitutions do not influence the structural stability of the enzyme in assays performed in vitro. Thermal inactivation of Rubisco purified from wild type (○) and the D470P/T471A/I472M/K474T quadruple mutant (●). Rubisco enzymes were incubated at each temperature for 10 min, and then assayed for RuBP carboxylase activity at 25°C. Activities were normalized to the specific activities measured after the 35°C incubation (wild type, 1.7 μmol/min/mg; D470P/T471A/I472M/K474T, 1.2 μmol/min/mg).
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Figure 3: Carboxy-terminal substitutions do not influence the structural stability of the enzyme in assays performed in vitro. Thermal inactivation of Rubisco purified from wild type (○) and the D470P/T471A/I472M/K474T quadruple mutant (●). Rubisco enzymes were incubated at each temperature for 10 min, and then assayed for RuBP carboxylase activity at 25°C. Activities were normalized to the specific activities measured after the 35°C incubation (wild type, 1.7 μmol/min/mg; D470P/T471A/I472M/K474T, 1.2 μmol/min/mg).

Mentions: To assess the effect of the mutant substitutions on holoenzyme levels in vivo, protein extract was subjected to SDS-PAGE and western-blot analysis. As shown in Fig. 2, the D470P/T471A/I472M/K474T quadruple-mutant strain contains wild-type levels of Rubisco subunits. A wild-type level of holoenzyme could also be purified from the quadruple mutant when cell extract was fractionated on a sucrose-density gradient (data not shown). No difference was detected between the mutant and wild-type enzymes when thermal stability experiments were performed in vitro (Fig. 3). Thus, analysis of the catalytic properties of the quadruple-mutant enzyme would not be expected to be influenced by structural instability during the assays.


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

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

Carboxy-terminal substitutions do not influence the structural stability of the enzyme in assays performed in vitro. Thermal inactivation of Rubisco purified from wild type (○) and the D470P/T471A/I472M/K474T quadruple mutant (●). Rubisco enzymes were incubated at each temperature for 10 min, and then assayed for RuBP carboxylase activity at 25°C. Activities were normalized to the specific activities measured after the 35°C incubation (wild type, 1.7 μmol/min/mg; D470P/T471A/I472M/K474T, 1.2 μmol/min/mg).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Carboxy-terminal substitutions do not influence the structural stability of the enzyme in assays performed in vitro. Thermal inactivation of Rubisco purified from wild type (○) and the D470P/T471A/I472M/K474T quadruple mutant (●). Rubisco enzymes were incubated at each temperature for 10 min, and then assayed for RuBP carboxylase activity at 25°C. Activities were normalized to the specific activities measured after the 35°C incubation (wild type, 1.7 μmol/min/mg; D470P/T471A/I472M/K474T, 1.2 μmol/min/mg).
Mentions: To assess the effect of the mutant substitutions on holoenzyme levels in vivo, protein extract was subjected to SDS-PAGE and western-blot analysis. As shown in Fig. 2, the D470P/T471A/I472M/K474T quadruple-mutant strain contains wild-type levels of Rubisco subunits. A wild-type level of holoenzyme could also be purified from the quadruple mutant when cell extract was fractionated on a sucrose-density gradient (data not shown). No difference was detected between the mutant and wild-type enzymes when thermal stability experiments were performed in vitro (Fig. 3). Thus, analysis of the catalytic properties of the quadruple-mutant enzyme would not be expected to be influenced by structural instability during the assays.

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