Functional hybrid rubisco enzymes with plant small subunits and algal large subunits: engineered rbcS cDNA for expression in chlamydomonas.

There has been much interest in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a target for engineering an increase in net CO(2) fixation in photosynthesis. Improvements in the enzyme would lead to an increase in the production of food, fiber, and renewable energy. Although the large subunit contains the active site, a family of rbcS nuclear genes encodes the Rubisco small subunits, which can also influence the carboxylation catalytic efficiency and CO(2)/O(2) specificity of the enzyme. To further define the role of the small subunit in Rubisco function, small subunits from spinach, Arabidopsis, and sunflower were assembled with algal large subunits by transformation of a Chlamydomonas reinhardtii mutant that lacks the rbcS gene family. Foreign rbcS cDNAs were successfully expressed in Chlamydomonas by fusing them to a Chlamydomonas rbcS transit peptide sequence engineered to contain rbcS introns. Although plant Rubisco generally has greater CO(2)/O(2) specificity but a lower carboxylation V(max) than Chlamydomonas Rubisco, the hybrid enzymes have 3-11% increases in CO(2)/O(2) specificity and retain near normal V(max) values. Thus, small subunits may make a significant contribution to the overall catalytic performance of Rubisco. Despite having normal amounts of catalytically proficient Rubisco, the hybrid mutant strains display reduced levels of photosynthetic growth and lack chloroplast pyrenoids. It appears that small subunits contain the structural elements responsible for targeting Rubisco to the algal pyrenoid, which is the site where CO(2) is concentrated for optimal photosynthesis.This work was supported in part by Grant DE-FG02-00ER15044 from the United States Department of Energy

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

<p>Abstract</p> <p>Background</p> <p>Ribulose-1,5-bisphosphate is the rate-limiting enzyme in photosynthesis. The catalytic large subunit of the green-algal enzyme from <it>Chlamydomonas reinhardtii </it>is ~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 <it>Chlamydomonas </it>large subunit to mimic the sequence of higher-specificity plant enzymes.</p> <p>Results</p> <p>The quadruple-mutant enzyme has a 10% increase in CO₂/O₂specificity and a lower carboxylation catalytic efficiency. The mutations do not seem to influence the protein expression, structural stability or the function in vivo.</p> <p>Conclusion</p> <p>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.</p

Functional Hybrid Rubisco Enzymes with Plant Small Subunits and Algal Large Subunits \u3ci\u3eENGINEERED rbcS cDNA FOR EXPRESSION IN CHLAMYDOMONAS\u3c/i\u3e

There has been much interest in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a target for engineering an increase in net CO2 fixation in photosynthesis. Improvements in the enzyme would lead to an increase in the production of food, fiber, and renewable energy. Although the large subunit contains the active site, a family of rbcS nuclear genes encodes the Rubisco small subunits, which can also influence the carboxylation catalytic efficiency and CO2/O2 specificity of the enzyme. To further define the role of the small subunit in Rubisco function, small subunits from spinach, Arabidopsis, and sunflower were assembled with algal large subunits by transformation of a Chlamydomonas reinhardtii mutant that lacks the rbcS gene family. Foreign rbcS cDNAs were successfully expressed in Chlamydomonas by fusing them to a Chlamydomonas rbcS transit peptide sequence engineered to contain rbcS introns. Although plant Rubisco generally has greater CO2/O2 specificity but a lower carboxylation Vmax than Chlamydomonas Rubisco, the hybrid enzymes have 3–11% increases inCO2/O2 specificity and retain near normal Vmax values. Thus, small subunits may make a significant contribution to the overall catalytic performance of Rubisco. Despite having normal amounts of catalytically proficient Rubisco, the hybrid mutant strains display reduced levels of photosynthetic growth and lack chloroplast pyrenoids. It appears that small subunits contain the structural elements responsible for targeting Rubisco to the algal pyrenoid, which is the site where CO2 is concentrated for optimal photosynthesis

Reduced CO\u3csub\u3e2\u3c/sub\u3e/O\u3csub\u3e2\u3c/sub\u3e specificity of ribulose-bisphosphate carboxylase/oxygenase in a temperature-sensitive chloroplast mutant of \u3ci\u3eChlamydomonas\u3c/i\u3e

The Chlamydomonas reinhardtii chloroplast mutant 68-4PP is phenotypically indistinguishable from wild type at 25°C but fails to grow photosynthetically at 35°C. It had about 30% of the wild-type level of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) holoenzyme and carboxylase activity when grown at 25°C, but less than 15% when grown at 35°C. Pulse-labeling with 35S showed that the decrease in enzyme level at the restrictive temperature was not a result of reduced synthesis of enzyme subunits. The CO2/O2 specificity factor (VcKo/VoKc, where Vc and Vo are Vmax values for carboxylation and oxygenation and Kc and Ko are Km values for CO2 and 02) of the mutant enzyme was found to be significantly less than that of the wild-type enzyme (54 ± 2 and 62 ± 1, respectively), and this alteration was accompanied by increases in Ko and Kc and a decrease in Vc/Vo. DNA sequencing revealed a single missense mutation in the 684PP chloroplast large-subunit gene. This mutation causes leucine to be replaced by phenylalanine at position 290 in the large subunit polypeptide sequence. These results (i) support previous studies that implicated this region of the large subunit as an important structural component of the enzyme\u27s function and (ii) demonstrate that chloroplast genetic modification of the CO2/O2 specificity factor of a plant-type carboxylase/oxygenase is feasible

Reduced CO\u3csub\u3e2\u3c/sub\u3e/O\u3csub\u3e2\u3c/sub\u3e specificity of ribulose-bisphosphate carboxylase/oxygenase in a temperature-sensitive chloroplast mutant of \u3ci\u3eChlamydomonas\u3c/i\u3e

The Chlamydomonas reinhardtii chloroplast mutant 68-4PP is phenotypically indistinguishable from wild type at 25°C but fails to grow photosynthetically at 35°C. It had about 30% of the wild-type level of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) holoenzyme and carboxylase activity when grown at 25°C, but less than 15% when grown at 35°C. Pulse-labeling with 35S showed that the decrease in enzyme level at the restrictive temperature was not a result of reduced synthesis of enzyme subunits. The CO2/O2 specificity factor (VcKo/VoKc, where Vc and Vo are Vmax values for carboxylation and oxygenation and Kc and Ko are Km values for CO2 and 02) of the mutant enzyme was found to be significantly less than that of the wild-type enzyme (54 ± 2 and 62 ± 1, respectively), and this alteration was accompanied by increases in Ko and Kc and a decrease in Vc/Vo. DNA sequencing revealed a single missense mutation in the 684PP chloroplast large-subunit gene. This mutation causes leucine to be replaced by phenylalanine at position 290 in the large subunit polypeptide sequence. These results (i) support previous studies that implicated this region of the large subunit as an important structural component of the enzyme\u27s function and (ii) demonstrate that chloroplast genetic modification of the CO2/O2 specificity factor of a plant-type carboxylase/oxygenase is feasible

Principles and principals: Do customer stewardship and agency control compete or complement when shaping frontline employee behavior?

This article introduces customer stewardship control (CSC) to the marketing field. This concept represents a frontline employee's felt ownership of and moral responsibility for customers' overall welfare. In two studies, the authors show that CSC is a more encompassing construct than customer orientation, which reflects a frontline employee's focus on meeting customers' needs. They provide evidence that the former is more potent in shaping in- and extra-role employee behaviors. Moreover, they highlight how CSC operates in conjunction with an organization's agency control system: Stewardship's positive influence on in- and extra-role behavior is weaker in the presence of high agency control. They offer actionable advice about how to solve the resulting managerial control dilemma. Finally, the authors show that CSC depends on drivers that reside at the individual level (employee relatedness), the team level (team competence), or both levels of aggregation (employee and team autonomy). These findings show how to effectively design a frontline employee's work environment to ensure optimal frontline performance

Measuring the Intrapersonal Component of Psychological Empowerment: Confirmatory Factor Analysis of the Sociopolitical Control Scale

The Sociopolitical Control Scale (SPCS) is a widely used measure of the intrapersonal component of psychological empowerment. Confirmatory factor analyses (CFA) were conducted with data from two samples to test the hypothesized structure of the SPCS, the potential effects of method bias on the measure's psychometric properties, and whether a revised version of the scale (SPCS‐R) yielded improved model fit. Sample 1 included 316 randomly selected community residents of the Midwestern United States. Sample 2 included 750 community residents of the Northeastern U.S. Results indicated that method bias from the use of negatively worded items had a significant effect on the factor structure of the SPCS. CFA of the SPCS‐R, in which negatively worded items were rephrased so that all statements were positively worded, supported the measure's hypothesized two‐factor structure (i.e., leadership competence and policy control). Subscales of the SPCS‐R were found reliable and related in expected ways with measures of community involvement. Implications of the study for empowerment‐based research and practice are described, and strategies to further develop the SPCS are discussed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/117223/1/ajcp9070.pd

Directive versus empowering leadership: A field experiment comparing impacts on task proficiency and proactivity

Using a field experiment in the United Arab Emirates, we compared the impacts of directive and empowering leadership on customer-rated core task proficiency and proactive behaviors. Results of tests for main effects demonstrated that both directive and empowering leadership increased work unit core task proficiency, but only empowering leadership increased proactive behaviors. Examination of boundary conditions revealed that directive leadership enhanced proactive behaviors for work units that were highly satisfied with their leaders, whereas empowering leadership had stronger effects on both core task proficiency and proactive behaviors for work units that were less satisfied with their leaders. We discuss implications for both theory and practice. © Academy of Management Journal