Adaptation of photosynthesis in marama bean Tylosema esculentum (Burchell A. Schreiber) to a high temperature, high radiation, drought-prone environment.

Marama bean, Tylosema esculentum, is a tuberous legume native to the Kalahari region of Southern Africa where it grows under high temperatures (typical daily max 37 degrees C during growing season) and radiation (frequently in excess of 2000 micromol m(-2) s(-1)) in sandy soils with low rainfall. These conditions might be expected to select for increased water-use efficiency of photosynthesis. However, marama was found to give similar leaf photosynthetic rates to other C3 plants for a given internal leaf CO2 concentration and Rubisco content. Under conditions of increasing drought, no increase in water-use efficiency of photosynthesis was observed, but stomata closed early and preceded any change in leaf water potential. The possibility of subtle adaptations of photosynthetic characteristics to its natural environment were investigated at the level of Rubisco kinetics. The specificity factor of marama Rubisco was slightly lower than that of wheat, but the apparent Km for CO2 in air (Km') was about 20% lower than that of wheat. This is consistent with better adaptation for efficient photosynthesis at high temperatures in marama compared to wheat, although the net benefit is predicted to be very small (<0.5% at 35 degrees C). The sequence of marama rbcL gene shows 27 deduced amino acid residue differences from that for wheat, and the possibility that one or more of these cause the difference in Rubisco Km' is discussed.

beta-lactamase I from Bacillus cereus. Structure and site-directed mutagenesis.

The sequence of the gene for beta-lactamase I from Bacillus cereus 569/H has been redetermined. Oligonucleotide-directed mutagenesis has been carried out, and the effects of the changes on the ampicillin-resistance of Escherichia coli TG1 expressing the mutant genes have been studied. Lysine-73, close to the active-site serine-70 and a highly-conserved residue, has been converted into arginine. This change had a large effect on activity, but did not abolish it. An even larger effect was found in the mutant in which glutamate-166 had been converted into glutamine; this had little or no activity. On the other hand, the conversion of glutamate-168 into aspartate gave fully active enzyme. Glutamate-166 is an invariant residue, but glutamate-168 is not. Alanine-123 has been replaced by cysteine, to give active enzyme; this change forms part of the plan to introduce a disulphide bond into the enzyme.

Effect of mutations of residue 340 in the large subunit polypeptide of Rubisco from Anacystis nidulans.

Residues 338-342 at the C-terminal end of loop 6 in the large subunit beta/alpha barrel structure of Rubisco influence specificity towards CO2 and O2. In Anacystis nidulans Rubisco, replacement of alanine 340 by tyrosine or histidine increased the specificity factor by 12-13%, accompanied by a 25-33% fall in Vc, the rate of carboxylation, while replacement by asparagine increased the specificity factor by 9% and Vc by 19%. Other mutations did not significantly alter specificity. Alanine 340 does not interact directly with the bisphosphate substrate, thus replacing it with other residues must have indirect effects on the specificity factor and rate of carboxylation.

Effect of mutation of lysine-128 of the large subunit of ribulose bisphosphate carboxylase/oxygenase from Anacystis nidulans.

The contribution of lysine-128 within the active site of Anacystis nidulans d-ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) was investigated by the characterization of mutants in which lysine-128 was replaced with arginine, glycine, glutamine, histidine or aspartic acid. Mutated genes encoding the Rubisco large subunit were expressed in Escherichia coli and the resultant polypeptides assembled into active complexes. All of the mutant enzymes had a lower affinity for ribulose 1,5-bisphosphate (RuBP) and lower rates of carboxylation. Substitution of lysine-128 with glutamine, histidine or aspartic acid decreased the specificity factor and led to the production of an additional monophosphate reaction product. We show that this product results from the loss of the phosphate from C-1 of RuBP, most probably by beta-elimination from the 2,3-enediolate derivative of RuBP. The results confirm that lysine-128 is important in determining the position of the essential epsilon-amino group of lysine-334 within the active site and in loop dynamics. This further demonstrates that residues remote from the active site can be manipulated to modify catalytic function.

Manipulation of Rubisco: the amount, activity, function and regulation.

Genetic modification to increase the specificity of Rubisco for CO(2) relative to O(2) and to increase the catalytic rate of Rubisco in crop plants would have great agronomic importance. The availability of three-dimensional structures of Rubisco at atomic resolution and the characterization of site-directed mutants have greatly enhanced the understanding of the catalytic mechanism of Rubisco. Considerable progress has been made in identifying natural variation in the catalytic properties of Rubisco from different species and in developing the tools for introducing both novel and foreign Rubisco genes into plants. The additional complexities of assembling copies of the two distinct polypeptide subunits of Rubisco into a functional holoenzyme in vivo (requiring sufficient expression, post-translational modification, interaction with chaperonins, and interaction with Rubisco activase) remain a major challenge. The consequences of changing the amount of Rubisco present in leaves have been investigated by the use of antisense constructs. The manipulation of genes encoding Rubisco activase has provided a means to investigate the regulation of Rubisco activity.