Regulation of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activation by Inorganic Phosphate through Stimulating the Binding of the Activator CO2 to the Activation Sites

The mechanism of the regulation of the activation of ribulose1,5-bisphosphate carboxylase/ oxygenase (RuBisCO) by inorganicphosphate (P_i) in the presence of limiting concentrations ofCO₂ was explored. The activation state of RuBisCO increasedsigmoidally following a biphasic kinetics against the concentrationof P_i in the activation mixture with an intermediary plateauat 2 to 3 mM P_i when the enzyme was activated for 30 min. Theintermediary plateau could not be seen when the preincubationtime was 10 min and the activation was completed at 10 mM P_i.RuBisCO from Euglena also showed a quite similar activationkinetics. The activation was not due to the contaminating CO₂included in the stock P_i solution or in the activation buffercontaining the enzyme. The experiments with 2-carboxyarabinitol1,5-bisphosphate showed that the P_i stimulated activation wasdue to the promotion of binding of the activator CO₂ to theactivation sites. It was also found that P_i increased the affinityof RuBisCO for the activator CO₂ 5.4-fold accompanied by a decreaseof the half-saturating concentration of CO₂ to 1.6 µMat 20 mM MgCl₂. Physiological significance of the effects ofP_i on the activation of RuBisCO is discussed. ²Present address: Laboratory of Plant Molecular Physiology,Research Institute of Innovative Technology for the Earth (RITE),9-2 Kizugawadai, Kizu-cho, Soraku-gun, Kyoto, Japan.     

Genomic analysis of mutants affecting xanthophyll biosynthesis and regulation of photosynthetic light harvesting in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

When the absorption of light energy exceeds the capacity for its utilization in photosynthesis, regulation of light harvesting is critical in order for photosynthetic organisms to minimize photo-oxidative damage. Thermal dissipation of excess absorbed light energy, measured as non-photochemical quenching (NPQ) of chlorophyll fluorescence, is induced rapidly in response to excess light conditions, and it is known that xanthophylls such as zeaxanthin and lutein, the transthylakoid pH gradient, and the PsbS protein are involved in this mechanism. Although mutants affecting NPQ and the biosynthesis of zeaxanthin and lutein were originally isolated and characterized at the physiological level in the unicellular green alga Chlamydomonas reinhardtii, the molecular basis of several of these mutants, such as npq1 and lor1, has not been determined previously. The recent sequencing of the C. reinhardtii nuclear genome has facilitated the search for C. reinhardtii homologs of plant genes involved in xanthophyll biosynthesis and regulation of light harvesting. Here we report the identification of C. reinhardtii genes encoding PsbS and lycopene ɛ-cyclase, and we show that the lor1 mutation, which affects lutein synthesis, is located within the lycopene ɛ-cyclase gene. In contrast, no homolog of the plant violaxanthin de-epoxidase (VDE) gene was found. Molecular markers were used to map the npq1 mutation, which affects VDE activity, as a first step toward the map-based cloning of the NPQ1 gene.     

Activation of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase by Inorganic Phosphate under Nocturnal Conditions

In vivo activation states of ribulose 1,5-bisphosphate carboxylase/oxygenase(RuBisCO; EC 4.1.1.39 [EC] ) in the dark and light phases were measuredin intact leaves of Phaseolus and radish. The activation statewas high in the dark and comparable to the activation stateunder illumination at saturating light intensity. Then, we examined,using RuBisCO purified from spinach leaves, a mechanism forthe activation of RuBisCO in the dark when the stroma is neutralizedand lossess Mg²⁺ partly. Activation was not obserevd when theenzyme was incubated at air-level CO₂ and 10 mM Mg²⁺ at pH rangingfrom 6.2 to 7.5. However, the activation was highly promotedin this pH range when the activation mixture contained 10 mMinorganic phosphate. The activation state was 50 to 60% betweenpH 7.0 and 7.8 and maximum over pH 8.2 in the presence of 10mM inorganic phosphate. Studies of the initial rate of activationshow that the promotion of activation was through stabilizationof the active form of the enzyme by inorganic phosphate, notby altering the pKa of the activator -amino group of Lys-201.The physiological significance of the activation of RuBisCOby inorganic phosphate in the dark is discussed. ³ Present address: Department of Biochemistry, University ofNebraska, Lincoln, NE 68588-0664, U.S.A.     

Chemical modification of arginine alleviates the decline in activity during catalysis of spinach Rubisco

Arginine residues of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were chemically modified with phenylglyoxal (PhG). PhG inactivated Rubisco with a half-time of 20-25 min. An inclusion of a catalytic product, 3-phosphoglycerate (PGA), protected Rubisco from inactivation and delayed the half-time to 60-90 min. Peptide mapping and sequencing of Rubisco modified for 60 min with radiolabeled PhG in the presence of 10mM PGA revealed that Arg187, Arg258, and Arg431 of the large subunit were modified. The extent and rate of the decline in activity during catalysis (fallover phenomenon) were reduced by the modification. This is the first report identifying PhG-modified arginine residues and to demonstrate the effect of the modification of arginine residues on the kinetics of fallover.     

Chimeric small subunits influence catalysis without causing global conformational changes in the crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase

Comparison of subunit sequences and X-ray crystal structures of ribulose-1,5-bisphosphate carboxylase/oxygenase indicates that the loop between beta-strands A and B of the small subunit is one of the most variable regions of the holoenzyme. In prokaryotes and nongreen algae, the loop contains 10 residues. In land plants and green algae, the loop is comprised of approximately 22 and 28 residues, respectively. Previous studies indicated that the longer betaA-betaB loop was required for the assembly of cyanobacterial small subunits with plant large subunits in isolated chloroplasts. In the present study, chimeric small subunits were constructed by replacing the loop of the green alga Chlamydomonas reinhardtii with the sequences of Synechococcus or spinach. When these engineered genes were transformed into a Chlamydomonas mutant that lacks small-subunit genes, photosynthesis-competent colonies were recovered, indicating that loop size is not essential for holoenzyme assembly. Whereas the Synechococcus loop causes decreases in carboxylation V(max), K(m)(O(2)), and CO(2)/O(2) specificity, the spinach loop causes complementary decreases in carboxylation V(max), K(m)(O(2)), and K(m)(CO(2)) without a change in specificity. X-ray crystal structures of the engineered proteins reveal remarkable similarity between the introduced betaA-betaB loops and the respective loops in the Synechococcus and spinach enzymes. The side chains of several large-subunit residues are altered in regions previously shown by directed mutagenesis to influence CO(2)/O(2) specificity. Differences in the catalytic properties of divergent Rubisco enzymes may arise from differences in the small-subunit betaA-betaB loop. This loop may be a worthwhile target for genetic engineering aimed at improving photosynthetic CO(2) fixation.     

Deletion of nine carboxy-terminal residues of the Rubisco small subunit decreases thermal stability but does not eliminate function

A recent X-ray crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from the green alga Chlamydomonas reinhardtii lacks 13 carboxy-terminal residues of the small subunit. To determine the importance of this divergent region, a non-sense mutation was created that removes nine residues. This engineered gene was transformed into a Chlamydomonas strain that lacks the small-subunit gene family. The resulting holoenzyme has a normal CO₂/O₂ specificity but decreased carboxylation V_max. Whereas wild-type enzyme retained most of its carboxylase activity after a 10-min incubation at 55°C, the mutant enzyme was inactivated. Thus, although disordered or divergent, the carboxy terminus is required for maximal activity and stability.     

The role of structural intersubunit microheterogeneity in the regulation of the activity in hysteresis of ribulose 1, 5-bisphosphate carboxylase/oxygenase

Many enzymes are composed of subunits with the identical primary structure. It has been believed that the protein structure of these subunits is the same. Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) comprises eight large subunits with the identical amino acid sequence and eight small subunits. Rotation of the side chains of the lysine residues, Lys-21 and Lys-305, in each of the eight large subunits in spinach RuBisCO in two ways produces microheterogeneity among the subunits. These structures are stabilized through hydrogen bonds by water molecules incorporated into the large subunits. This may cause different effects upon catalysis and a hysteretic, time-dependent decrease in activity in spinach RuBisCO. Changing the amino acid residues corresponding to Lys-21 and Lys-305 in non-hysteretic Chromatium vinosum RuBisCO to lysine induces hysteresis and increases the catalytic activity from 8.8 to 15.8 per site per second. This rate is approximately five times higher than that of the higher-plant enzyme.     

Application of machine learning methods to pathogen safety evaluation in biological manufacturing processes

The production of recombinant therapeutic proteins from animal or human cell lines entails the risk of endogenous viral contamination from cell substrates and adventitious agents from raw materials and environment. One of the approaches to control such potential viral contamination is to ensure the manufacturing process can adequately clear the potential viral contaminants. Viral clearance for production of human monoclonal antibodies is achieved by dedicated unit operations, such as low pH inactivation, viral filtration, and chromatographic separation. The process development of each viral clearance step for a new antibody production requires significant effort and resources invested in wet laboratory experiments for process characterization studies. Machine learning methods have the potential to help streamline the development and optimization of viral clearance unit operations for new therapeutic antibodies. The current work focuses on evaluating the usefulness of machine learning methods for process understanding and predictive modeling for viral clearance via a case study on low pH viral inactivation.