Structural and molecular basis of starch viscosity in hexaploid wheat.

Wheat starch is considered to have a low paste viscosity relative to other starches. Consequently, wheat starch is not preferred for many applications as compared to other high paste viscosity starches. Increasing the viscosity of wheat starch is expected to increase the functionality of a range of wheat flour-based products in which the texture is an important aspect of consumer acceptance (e.g., pasta, and instant and yellow alkaline noodles). To understand the molecular basis of starch viscosity, we have undertaken a comprehensive structural and rheological analysis of starches from a genetically diverse set of wheat genotypes, which revealed significant variation in starch traits including starch granule protein content, starch-associated lipid content and composition, phosphate content, and the structures of the amylose and amylopectin fractions. Statistical analysis highlighted the association between amylopectin chains of 18-25 glucose residues and starch pasting properties. Principal component analysis also identified an association between monoesterified phosphate and starch pasting properties in wheat despite the low starch-phosphate level in wheat as compared to tuber starches. We also found a strong negative correlation between the phosphate ester content and the starch content in flour. Previously observed associations between internal starch granule fatty acids and the swelling peak time and pasting temperature have been confirmed. This study has highlighted a range of parameters associated with increased starch viscosity that could be used in prebreeding/breeding programs to modify wheat starch pasting properties.

Gene expression in a starch synthase IIa mutant of barley: changes in the level of gene transcription and grain composition.

The barley shrunken grain mutant M292 has a novel high-amylose starch phenotype caused by a mutation in the starch synthase IIa gene (SsIIa) located at the starch excess-6 (sex6) locus on chromosome 7H of barley. The loss of SSIIa enzyme activity leads to a decrease in amylopectin synthesis to less than 20% of the levels found in wild-type grains. Detailed composition analysis indicates that the contents of protein, non-starch polysaccharides, lipid, sucrose and hexoses, and fructo-oligosaccharides are increased in mature M292 grain compared to wild type. Using a microarray analysis, we characterize the differences between the transcription profiles of wild-type and mutant barley endosperms at mid-grain fill. The expression changes include genes involved in carbon storage, stress-related genes, and a number of transcripts with unassigned function. The changes in gene expression are discussed in terms of the altered grain composition of the mutant seed.

Genetic characterisation of dough rheological properties in a wheat doubled haploid population: additive genetic effects and epistatic interactions.

Doubled haploid lines (n = 160) from a cross between wheat cultivars 'Cranbrook' (high dough extensibility) and 'Halberd' (low dough extensibility) were grown at three Australian locations. The parents differ at all high- and low-molecular-weight glutenin loci. Dough rheological parameters were measured using small-scale testing procedures, and quantitative trait locus (QTL) mapping procedures were carried out using an existing well-saturated genetic linkage map for this cross. Genetic parameters were estimated using three software packages: QTLCartographer, Epistat and Genstat. Results indicated that environmental factors are a major determinant of dough extensibility across the three trial sites, whereas genotypic factors are the major determinants of dough strength. Composite interval mapping analysis across the 21 linkage groups revealed that as expected, the main additive QTLs for dough rheological properties are located at the high- and low-molecular-weight glutenin loci. A new QTL on chromosome 5A for M-extensibility (a mixograph-estimated measure of extensibility) was detected. Analysis of epistatic interactions revealed that there were significant conditional epistatic interactions related with the additive effects of glutenin loci on dough rheological properties. Therefore, the additive genetic effects of glutenins on dough rheological properties are conditional upon the genetic background of the wheat line. The molecular basis of the interactions with the glutenin loci may be via proteins that modify or alter the gluten protein matrix or variations in the expression level of the glutenin genes. Reverse-phase high performance liquid chromatography analysis of the molar number of individual glutenin subunits across the population showed that certain conditional epistases resulted in increased expression of the affected glutenin. The epistatic interactions detected in this study provide a possible explanation of the variable genetic effects of some glutenins on quality attributes in different genetic backgrounds and provide essential information for the accurate prediction of glutenin related variance in marker-assisted wheat breeding.

Characterization of low-molecular-weight glutenin genes in Aegilops tauschii.

This paper reports the characterization of the low-molecular-weight (LMW) glutenin gene family of Aegilops tauschii (syn. Triticum tauschii), the D-genome donor of hexaploid wheat. By analysis of bacterial artificial chromosome (BAC) clones positive for hybridization with an LMW glutenin probe, seven unique LMW glutenin genes were identified. These genes were sequenced, including their untranslated 3' and 5' flanking regions. The deduced amino acid sequences of the genes revealed four putative active genes and three pseudogenes. All these genes had a very high level of similarity to LMW glutenins characterized in hexaploid wheat. The predicted molecular weights of the mature proteins were between 32.2 kDa and 39.6 kDa, and the predicted isoelectric points of the proteins were between 7.53 and 8.06. All the deduced proteins were of the LMW-m type. The organization of the seven LMW glutenin genes appears to be interspersed over at least several hundred kilo base pairs, as indicated by the presence of only one gene or pseudogene per BAC clone. Southern blot analysis of genomic DNA of Ae. tauschii and the BAC clones containing the seven LMW glutenin genes indicated that the BAC clones contained all LMW glutenin-hybridizing bands present in the genome. Two-dimensional gel electrophoresis of an LMW glutenin extract from Ae. tauschii was conducted and showed the presence of at least 11 distinct proteins. Further analysis indicated that some of the observed proteins were modified gliadins. These results suggest that the actual number of typical LMW glutenins may in fact be much lower than previously thought, with a number of modified gliadins also being present in the polymeric fraction.

Molecular discrimination of Bx7 alleles demonstrates that a highly expressed high-molecular-weight glutenin allele has a major impact on wheat flour dough strength.

High-molecular-weight glutenin subunits (HMW-GS) are important determinants of wheat dough quality as they confer visco-elastic properties to the dough required for mixing and baking performance. With this important role, the HMW-GS alleles are key markers in breeding programs. In this work, we present the use of a PCR marker initially designed to discriminate Glu1 Bx7 and Glu1 Bx17 HMW-GS. It was discovered that this marker also differentiated two alleles, originally both scored as Glu1 Bx7, present in the wheat lines CD87 and Katepwa respectively, by a size polymorphism of 18 bp. The marker was scored across a segregating doubled-haploid (DH) population (CD87 x Katepwa) containing 156 individual lines and grown at two sites. Within this population, the marker differentiated lines showing the over-expression of the Glu1 Bx7 subunit (indicated by the larger PCR fragment), derived from the CD87 parent, relative to lines showing the normal expression of the Glu1 Bx7 subunit, derived from the Katepwa parent. DNA sequence analysis showed that the observed size polymorphism was due to an 18 bp insertion/deletion event at the C-terminal end of the central repetitive domain of the Glu1 Bx 7 coding sequence, which resulted in an extra copy of the hexapeptide sequence QPGQGQ in the deduced amino-acid sequence of Bx7 from CD87. When the DH population was analysed using this novel Bx7 PCR marker, SDS PAGE and RP HPLC, there was perfect correlation between the Bx7 PCR marker results and the expression level of Bx7. This differentiation of the population was confirmed by both SDS-PAGE and RP-HPLC. The functional significance of this marker was assessed by measuring key dough properties of the 156 DH lines. A strong association was shown between lines with an over expression of Bx7 and high dough strength. Furthermore, the data demonstrated that there was an additional impact of Glu-D1 alleles on dough properties, with lines containing both over-expressed Bx7 and Glu-D1 5+10 having the highest levels of dough strength. However, there was no statistically significant epistatic interaction between Glu-B1 and Glu-D1 loci.

The organization of genes tightly linked to the Ha locus in Aegilops tauschii, the D-genome donor to wheat.

The grain hardness locus, Ha, is located at the distal end of the short arm of chromosome 5D in wheat. Three polypeptides, puroindoline-a, puroindoline-b, and grain softness protein (GSP-1), have been identified as components of friabilin, a biochemical marker for grain softness, and the genes for these polypeptides are known to be tightly linked to the Ha locus. However, this region of the chromosome 5D has not been well characterized and the physical distance between the markers is not known. Separate lambda clones containing the puroindoline-a gene and the puroindoline-b gene have been isolated from an Aegilops tauschii (the donor of the D genome to wheat) genomic lambda library and investigated. Considerable variation appears to exist in the organization of the region upstream of the gene for puroindoline-b among species closely related to wheat. Using in situ hybridization the genes for puroindoline-a, -b, and GSP-1 were demonstrated to be physically located at the tip of the short arm of chromosome 5 of A. tauschii. Four overlapping clones were isolated from a large-insert BAC library constructed from A. tauschii and of these one contained genes for all of puroindoline-a, puroindoline-b, and GSP-1. The gene for puroindoline-a is located between the other two genes at a distance no greater than approximately 30 kb from either gene. The BAC clone containing all three known genes was used to screen a cDNA library constructed from hexaploid wheat and cDNAs that could encode novel polypeptides were isolated.

Two loci control phytoglycogen production in the monocellular green alga Chlamydomonas reinhardtii.

The STA8 locus of Chlamydomonas reinhardtii was identified in a genetic screen as a factor that controls starch biosynthesis. Mutations of STA8 cause a significant reduction in the amount of granular starch produced during nutrient limitation and accumulate phytoglycogen. The granules remaining in sta8 mutants are misshapen, and the abundance of amylose and long chains in amylopectin is altered. Mutations of the STA7 locus, which completely lack isoamylase activity, also cause accumulation of phytoglycogen, although sta8 and sta7 mutants differ in that there is a complete loss of granular starch in the latter. This is the first instance in which mutations of two different genetic elements in one plant species have been shown to cause phytoglycogen accumulation. An analytical procedure that allows assay of isoamylase in total extracts was developed and used to show that sta8 mutations cause a 65% reduction in the level of this activity. All other enzymes known to be involved in starch biosynthesis were shown to be unaffected in sta8 mutants. The same amount of total isoamylase activity (approximately) as that present in sta8 mutants was observed in heterozygous triploids containing two sta7 mutant alleles and one wild-type allele. This strain, however, accumulates normal levels of starch granules and lacks phytoglycogen. The total level of isoamylase activity, therefore, is not the major determinant of whether granule production is reduced and phytoglycogen accumulates. Instead, a qualitative property of the isoamylase that is affected by the sta8 mutation is likely to be the critical factor in phytoglycogen production.

Biochemical characterization of wild-type and mutant isoamylases of Chlamydomonas reinhardtii supports a function of the multimeric enzyme organization in amylopectin maturation.

Chlamydomonas reinhardtii mutants of the STA8 gene produce reduced amounts of high amylose starch and phytoglycogen. In contrast to the previously described phytoglycogen-producing mutants of C. reinhardtii that contain no residual isoamylase activity, the sta8 mutants still contained 35% of the normal amount of enzyme activity. We have purified this residual isoamylase and compared it with the wild-type C. reinhardtii enzyme. We have found that the high-mass multimeric enzyme has reduced its average mass at least by one-half. This coincides with the disappearance of two out of the three activity bands that can be seen on zymogram gels. Wild-type and mutant enzymes are shown to be located within the plastid. In addition, they both act by cleaving off the outer branches of polysaccharides with no consistent difference in enzyme specificity. Because the mutant enzyme was demonstrated to digest phytoglycogen to completion in vitro, we propose that its inability to do so in vivo supports a function of the enzyme complex architecture in the processing of pre-amylopectin chains.

Comparison of starch-branching enzyme genes reveals evolutionary relationships among isoforms. Characterization of a gene for starch-branching enzyme IIa from the wheat genome donor Aegilops tauschii.

Genes and cDNAs for starch-branching enzyme II (SBEII) have been isolated from libraries constructed from Aegilops tauschii and wheat (Triticum aestivum) endosperm, respectively. One class of genes has been termed wSBEII-DA1 and encodes the N terminus reported for an SBEII from wheat endosperm. On the basis of phylogenetic comparisons with other branching enzyme sequences, wSBEII-DA1 is considered to be a member of the SBEIIa class. The wSBEII-DA1 gene consists of 22 exons with exons 4 to 21 being identical in length to the maize (Zea mays) SBEIIb gene, and the gene is located in the proximal region of the long arm of chromosome 2 at a locus designated sbe2a. RNA encoding SBEIIa can be detected in the endosperm from 6 d after flowering and is at its maximum level from 15 to 18 d after anthesis. Use of antibodies specific for SBEIIa demonstrated that this protein was present in both the soluble and granule bound fractions in developing wheat endosperm. We also report a cDNA sequence for SBEIIa that could arise by variant transcription/splicing. A second gene, termed wSBEII-DB1, was isolated and encodes an SBEII, which shows greater sequence identity with SBEIIb-type sequences than with SBEIIa-type sequences. Comparisons of SBEII gene structures among wheat, maize, and Arabidopsis indicate the lineage of the SBEII genes.

The structure and expression of the wheat starch synthase III gene. Motifs in the expressed gene define the lineage of the starch synthase III gene family.

The endosperm of hexaploid wheat (Triticum aestivum [L.]) was shown to contain a high molecular weight starch synthase (SS) analogous to the product of the maize du1 gene, starch synthase III (SSIII; DU1). cDNA and genomic DNA sequences encoding wheat SSIII were isolated and characterized. The wheat SSIII cDNA is 5,346 bp long and contains an open reading frame that encodes a 1,628-amino acid polypeptide. A putative N-terminal transit peptide, a 436-amino acid C-terminal catalytic domain, and a central 470-amino acid SSIII-specific domain containing three regions of repeated amino acid similarity were identified in the wheat gene. A fourth region between the transit peptide and the SSIII-specific domain contains repeat motifs that are variable with respect to motif sequence and repeat number between wheat and maize. In dicots, this N-terminal region does not contain repeat motifs and is truncated. The gene encoding wheat SSIII, designated ss3, consists of 16 exons extending over 10 kb, and is located on wheat chromosome I. Expression of ss3 mRNA in wheat was detected in leaves, pre-anthesis florets, and from very early to middle stage of endosperm development. The entire N-terminal variable repeat region and the majority of the SSIII-specific domain are encoded on a single 2,703-bp exon. A gene encoding a class III SS from the Arabidopsis genome sequencing project shows a strongly conserved exon structure to the wheat ss3 gene, with the exception of the N-terminal region. The evolutionary relationships of the genes encoding monocot and dicot class III SSs are discussed.

The localization and expression of the class II starch synthases of wheat.

The starch granules of hexaploid wheat (Triticum aestivum) contain a group of three proteins known as SGP-1 (starch granule protein-1) proteins, which have apparent molecular masses of 100, 108, and 115 kD. The nature and role of these proteins has not been defined previously. We demonstrate that these polypeptides are starch synthases that are present in both the starch granule and the soluble fraction at the early stages of wheat endosperm development, but that are exclusively granule bound at mid and late endosperm development. A partial cDNA clone encoding a fragment of the 100-kD protein was obtained by screening a wheat endosperm cDNA expression library using monoclonal antibodies. Three classes of cDNA were subsequently isolated from a wheat endosperm cDNA library by nucleic acid hybridization and were shown to encode the 100-, 108-, and 115-kD proteins. The cDNA sequences are highly homologous to class II starch synthases and have the highest homology with the maize SSIIa (starch synthase IIa) gene. mRNA for the SGP-1 proteins was detected in the leaf, pre-anthesis florets, and endosperm of wheat and is highly expressed in the leaf and in the grain during the early to mid stages of development. We discuss the roles of the SGP-1 proteins in starch biosynthesis in wheat.

Analysis of starch structure using fluorophore-assisted carbohydrate electrophoresis.

The analysis of the fine structure of starches is important to the investigation of linkages between starch structure and function and to the investigation of the properties and roles of starch biosynthetic, modifying and degradation enzymes. Fluorophore-assisted carbohydrate electrophoresis has recently been introduced as a method for the analysis of the oligosaccharide populations released by the enzymatic digestion of starches, which has advantages in resolution and sensitivity over previously used methods, and provides the capacity for the facile analysis of oligosaccharide populations on either a molar or mass basis. The use of fluorophore-assisted carbohydrate electrophoresis for the analysis of oligosaccharides is reviewed with particular reference to the choice of label, efficiency of labeling and separation techniques. Examples of separations using slab gel electrophoresis, DNA sequencer analysis and capillary electrophoresis are presented and we conclude that on the basis of resolution and reproducibility, capillary electrophoresis is the method of choice for the separation of oligosaccharides of degree of polymerization from 1 to 100. Examples of isoamylase-debranched starches and glycogens analyzed by capillary electrophoresis are presented. The capillary electrophoresis analysis of starch structure through the analysis of oligosaccharides released by the debranching of limit dextrins derived from starches and glycogens is introduced as a useful diagnostic of starch structure. The potential for future development of novel diagnostics for starch structure using fluorophore-assisted carbohydrate electrophoresis is discussed.

Alpha-glucan binding of potato-tuber starch-branching enzyme I as determined by tryptophan fluorescence quenching, affinity electrophoresis and steady-state kinetics.

The affinity of potato tuber starch-branching enzyme-I (PSBE-I) for various linear malto-oligosaccharides, cyclodextrins, (CDs) and macromolecular alpha-glucans was investigated by alpha-glucan induced fluorescence quenching of intrinsic PSBE-I tryptophan residues and by affinity electrophoresis. alpha-Glucan binding was characterised by distinct shifts towards shorter wavelengths of the PSBE-I fluorescence emission spectrum and by concomitant reductions in fluorescence intensity. The magnitudes of both the maximum shift in emission spectrum and reduction in fluorescence intensity were dependent on the alpha-glucan ligands used. Maximum Kd for a range of linear malto-oligosaccharides analysed was 0.13 mM as found at a degree of polymerisation (DP) of 13. Large differences in dissociation constants were measured for CDs with DP 6 (alpha-CD, 6.0 mM), DP 7 (beta-CD, 0.25 mM) and DP 8 (gamma-CD, 0.67 microM). The high-molecular-mass alpha-glucans amylose and amylopectin, both substrates for PSBE-I, showed apparent affinities of 0.018 and 0.066 mg/ml, respectively. Small linear and cyclic oligosaccharides competed with amylopectin in the affinity electrophoresis system and they were also competitive inhibitors for PSBE-I activity. The affinities for oligosaccharides as measured by competition were, however, about 10-fold lower than as measured by fluorescence quenching suggesting the existence of a separate oligosaccharide binding site on PSBE-I. Affinity electrophoresis revealed multiform heterogeneity in the enzyme preparation with respect to alpha-glucan interaction.

Side reactions catalyzed by ribulose-bisphosphate carboxylase in the presence and absence of small subunits.

The large subunit core of ribulose-bisphosphate carboxylase from Synechococcus PCC 6301 expressed in Escherichia coli in the absence of its small subunits retains a trace of carboxylase activity (about 1% of the kcat of the holoenzyme) (Andrews, T. J (1988) J. Biol. Chem. 263, 12213-12219). During steady-state catalysis at substrate saturation, this residual activity diverted approximately 10% of the reaction flux to 1-deoxy-D-glycero-2,3-pentodiulose-5-phosphate as a result of beta elimination of inorganic phosphate from the first reaction intermediate, the 2,3-enediol form of ribulose bisphosphate. This indicates that the active site's ability to stabilize and/or retain this intermediate is compromised by the absence of small subunits. Epimerization and isomerization of the substrate resulting from misprotonation of the enediol intermediate were not significantly exacerbated by lack of small subunits. The residual carboxylating activity partitioned product between pyruvate and 3-phosphoglycerate in a ratio similar to that of the holoenzyme, indicating that stablization of the penultimate three-carbon aci-acid intermediate is not perturbed by lack of small subunits. The underlying instability of the five-carbon enediol intermediate was revealed, even with the holoenzyme, under conditions designed to lead to exhaustion of substrate CO2 (and O2). When carboxylation (and oxygenation) stalled upon exhaustion of gaseous substrate, both spinach and Synechococcus holoenzymes continued slowly to beta eliminate inorganic phosphate from and to misprotonate the enediol intermediate. With carboxylation and oxygenation blocked, the products of these side reactions of the enediol intermediate accumulated to readily detectable levels, illustrating the difficulties attendant upon ribulose-P2 carboxylase's use of this reactive species as a catalytic intermediate.

Differential expression and properties of starch branching enzyme isoforms in developing wheat endosperm.

Three forms of starch branching enzyme (BE) from developing hexaploid wheat (Triticum aestivum) endosperm have been partially purified and characterized. Immunological cross-reactivities indicate that two forms (WBE-IAD, 88 kD, and WBE-IB, 87 kD) are related to the maize BE I class and that WBE-II (88 kD) is related to maize BE II. Comparison of the N-terminal sequences from WBE-IAD and WBE-II with maize and rice BEs confirms these relationships. Evidence is presented from the analysis of nullisomic-tetrasomic wheat lines demonstrating that WBE-IB is located on chromosome 7B and that the WBE-IAD fraction contains polypeptides that are encoded on chromosomes 7A and 7D. The wheat endosperm BE classes are differentially expressed during endosperm development. WBE-II is expressed at a constant level throughout mid and late endosperm development. In contrast, WBE-IAD and WBE-IB are preferentially expressed in late endosperm development. Differences are also observed in the kinetic characteristics of the enzymes. The WBE-I isoforms have a 2- to 5-fold higher affinity for amylose than does WBE-II, and the WBE-I isoforms are activated up to 5-fold by phosphorylated intermediates and inorganic phosphate, whereas WBE-II is activated only 50%. The potential implications of this activation of BE I for starch biosynthesis are discussed.

High resolution slab gel electrophoresis of 8-amino-1,3, 6-pyrenetrisulfonic acid (APTS) tagged oligosaccharides using a DNA sequencer.

A novel electrophoretic method for the analysis of oligosaccharides using DNA sequencer technology is illustrated using malto-oligosaccharide distributions obtained following isoamylase digestion of glycogen, wheat starch and potato starch. The debranched starches were derivatized at the reducing and with the charged fluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS). This highly reproducible method provides baseline resolution of oligomers from chain lengths of 3 to more than 80 glucose units, and exhibits high sensitivity with detection thresholds of one femtomole per resolved band. In addition, the reductive amination procedure attaches a single fluorophore per oligosaccharide, allowing calculation of the results on either a mass or a molar basis. The efficacy of the method is illustrated through the determination of the profile of individual oligosaccharides of chain length with a degree of polymerization (DP) < 80, derived from loading less than 15 ng per analysis of glycogen, wheat and potato starches. While the results obtained were superior in resolution and sensitivity to previously reported observations using a range of techniques, they were nonetheless consistent with the overall differences between these polysaccharides. The resolution, sensitivity, reproducibility and high throughput of the method provides substantial advantages over existing methods for the analysis of linear oligosaccharide chain length distributions.

The isolation and characterisation of a gene encoding superoxide dismutase from Bradyrhizobium sp. (Parasponia) strain ANU289.

Bradyrhizobium sp. (Parasponia) strain ANU289 expresses a single Mn-SOD in both the vegetative and symbiotic states. A 500 bp sod-homologous sequence was amplified from genomic DNA of strain ANU289 using PCR. A 1.3 kb SalI fragment was subsequently cloned which contained an ORF, sodA, encoding a 23 Kd protein. This putative SOD shares considerable homology with other Mn-SODs and analysis of the sodA sequence predicts that it is expressed. A lacZ-sodA fusion complemented the SOD-deficiency of E. coli QC779 and resulted in the expression of SOD activity in both mutant and wild type E. coli. We conclude that sodA encodes the Mn-SOD of strain ANU289.

Mutations of an active site threonyl residue promote beta elimination and other side reactions of the enediol intermediate of the ribulosebisphosphate carboxylase reaction.

The side chain of residue threonine 65 within the active site of ribulosebisphosphate carboxylase participates in a network of hydrogen bonds and ionic interactions involving the phosphate moiety attached to C-1 of the substrate. This residue was replaced with serine, alanine, and valine in the enzyme from Synechococcus PCC 6301. The mutant enzymes were stable, expressed abundantly by Escherichia coli, and retained the ability to form gel-filterable complexes with the reaction-intermediate analog, 2'-carboxyarabinitol-1,5-bisphosphate. The substitutions reduced the kcat/Km(CO2) (where kcat is the substrate-saturated turnover rate) of the enzyme from 17- to 340-fold with the more radical substitutions causing more severe reductions. The CO2/O2 specificity also deteriorated progressively, the valine replacement causing a 2.3-fold reduction. In concert with these changes, a compound tentatively identified as 1-deoxy-D-glycero-2,3-pentodiulose-5-phosphate, the product of beta elimination of the 2,3-enediol(ate) intermediate of the catalytic reaction, appeared among the reaction products in progressively increasing amounts. In the case of the valine substitution, it comprised 13% of the ribulose bisphosphate consumed. The mutant enzymes also partitioned more of their reaction flux to pentulose bisphosphate isomers of ribulose bisphosphate. By contrast, the diversion of carboxylated product to pyruvate, as a result of beta elimination of the three-carbon aci-carbanion intermediate of the carboxylation reaction, was ameliorated by the replacements, the valine mutant showing a 5-fold improvement in this parameter. These observations focus attention on a geometric conflict which exists between the requirements for stabilization of the 5-carbon enediol(ate) and 3-carbon aci-carbanion intermediates. This conflict must be resolved by a change in the angle of the C-1/bridge oxygen bond during each catalytic cycle. The network of hydrogen bonds involving the side chain of threonine 65 must play a crucial role in facilitating reaction of the enediol(ate) with the gaseous substrate and in shepherding this subsequent movement.

Amino-terminal truncations of the ribulose-bisphosphate carboxylase small subunit influence catalysis and subunit interactions.

The first 20 residues at the amino terminus of the small subunit of spinach ribulose-1,5-bisphosphate carboxylase form an irregular arm that makes extensive contacts with the large subunit and also with another small subunit (S. Knight, I. Andersson, and C.-I. Brändén [1990] J Mol Biol 215: 113-160). The influence of these contacts on subunit binding and, indirectly, on catalysis was investigated by constructing truncations from the amino terminus of the small subunit of the highly homologous enzyme from Synechococcus PCC 6301 expressed in Escherichia coli. Removal of the first six residues (and thus the region of contact with a neighboring small subunit) affected neither the affinity with which the small subunits bound to the large subunits nor the catalytic properties of the assembled holoenzyme. Extending the truncation to include the first 12 residues (which encroaches into a highly conserved region that interacts with the large subunit) also did not weaken intersubunit binding appreciably, but it reduced the catalytic activity of the holoenzyme nearly 5-fold. Removal of an additional single residue (i.e. removal of a total of 13 residues) weakened intersubunit binding approximately 80-fold. Paradoxically, this partially restored catalytic activity to approximately 40% of that of the wild-type holoenzyme. None of these truncations materially affected the Km values for ribulose-1,5-bisphosphate or CO2. Removal of all 20 residues of the irregular arm (thereby deleting the conserved region of contact with large subunits) totally abolished the small subunit's ability to bind to large subunits to form a stable holoenzyme. However, this truncated small subunit was still synthesized by the E. coli cells. These data are interpreted in terms of the role of the amino-terminal arm of the small subunit in maintaining the structure of the holoenzyme.