Slow-binding inhibition of branching enzyme by the pseudooligosaccharide BAY e4609.

Branching enzyme from Escherichia coli is shown to be inhibited by the pseudooligosaccharide BAY e4609. The mechanism of binding is studied in detail by kinetics using reduced amylose as substrate. Lineweaver-Burk plots suggest the mechanism of a noncompetitive or slow-binding inhibitor. Further studies by progress curves and rate of loss of branching activity allows us to conclude BAY e4609 as being a slow-binding inhibitor of branching enzyme. We discuss how these results parallel the inhibition of alpha-amylase by acarbose and the significance of branching enzyme as belonging to the amylolytic family.

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis.

Incubation of maize branching enzyme, mBEI and mBEII, with 100 microM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants in E. coli showed a significant decrease of the activity and the mutant enzymes had Km values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.

Arginine residue 384 at the catalytic center is important for branching enzyme II from maize endosperm.

Branching enzyme (BE) belongs to the amylolytic family which contains four highly conserved regions. These regions are proposed to play an important role in catalysis as they are thought to be necessary for catalysis and/or binding the substrate. Only one arginine residue was found to be conserved in a catalytic center at the same position in all known sequences of BEs from various species as well as in the alpha-amylase enzyme family. In mBEII, a conserved Arg residue 384 is in catalytic region 2. We have used site-directed mutagenesis of the Arg-384 residue in order to study its possible role in BE. Previous chemical modification studies (H. Cao and J. Preiss, 1996, J. Prot. Chem. 15, 291-304) suggest that it may play a role in substrate binding. Replacement of Arg-384 by Ala, Ser, Gln, and Glu in the active site caused almost total inactivation. However, a conservative mutation of the conserved Arg-384 by Lys resulted in some residual activity, approximately 5% of the wild-type enzyme. The kinetics of the purified mutant R384K enzyme were investigated and no large effect on the Km of the substrate amylose for BE was observed. Thus, these results suggest that conserved Arg residue 384 in mBEII plays an important role in the catalytic function of BEs but may not be directly involved in substrate binding.

High-level expression of branching enzyme II from maize endosperm in Escherichia coli.

The gene that encodes the mature branching enzyme II (BEII) protein from maize (Zea mays L.) endosperm was amplified by means of a polymerase chain reaction technique and inserted into a T7-based expression vector. Although this has been an efficient expression system of maize BEII in Escherichia coli, an example is presented in this report which allows a greater expression of mBEII protein from the bacterial system by changing only one codon. The key to the level of expression appears to be related to the conversion of the third thymine base in the 285 position codon of the mBEII cDNA to cytosine without altering the encoded mBEII protein product. The crude cell extracts of enzyme prepared from E.coli exhibited seven-fold higher expression of branching enzyme activity compared to expression of the native enzyme. The enzymes from wild-type and the silent mutation genes were purified. The proteins were indistinguishable kinetically and immunologically. Thus, we obtained a significantly improved expression of mBEII protein in the bacterial system.

Preamylopectin Processing: A Mandatory Step for Starch Biosynthesis in Plants.

It has been generally assumed that the [alpha]-(1->4)-linked and [alpha]-(1->6)-branched glucans of starch are generated by the coordinated action of elongation (starch synthases) and branching enzymes. We have identified a novel Chlamydomonas locus (STA7) that when defective leads to a wipeout of starch and its replacement by a small amount of glycogen-like material. Our efforts to understand the enzymological basis of this phenotype have led us to determine the selective disappearance of an 88-kD starch hydrolytic activity. We further demonstrate that this enzyme is a debranching enzyme. Cleavage of the [alpha]-(1->6) linkage in a branched precursor of amylopectin (preamylopectin) has provided us with the ground rules for understanding starch biosynthesis in plants. Therefore, we propose that amylopectin clusters are synthesized by a discontinuous mechanism involving a highly specific glucan trimming mechanism.

Control of starch composition and structure through substrate supply in the monocellular alga Chlamydomonas reinhardtii.

In Chlamydomonas, as in higher plants, synthesis of ADP glucose catalyzed by ADP-glucose pyrophosphorylase is rate-limiting for the building of starch in the chloroplast. We have isolated disruptions of the STA1 ADP-glucose pyrophosphorylase structural gene that rendered the enzyme less responsive to the allosteric activator 3-phosphoglycerate. The structure and composition of the residual starch synthesized by all mutants of the STA1 locus is dramatically altered. The residual polysaccharide is shown to be devoid of amylose despite the presence of granule-bound starch synthase, the amylose biosynthetic enzyme. In addition, the fine structure of the mutant amylopectin revealed the presence of an altered chain-length distribution. This distribution mimicks that which is observed during growth and photosynthesis and differs markedly from that observed during storage. We therefore propose that low nucleotide sugar concentrations are either directly or indirectly responsible for the major differences observed in the composition or structure of starch during storage and photosynthesis.

Storage, Photosynthesis, and Growth: The Conditional Nature of Mutations Affecting Starch Synthesis and Structure in Chlamydomonas.

Growth-arrested Chlamydomonas cells accumulate a storage polysaccharide that bears strong structural and functional resemblance to higher plant storage starch. It is synthesized by similar enzymes and responds in an identical fashion to the presence of mutations affecting these activities. We found that log-phase photosynthetically active algae accumulate granular [alpha](1->4)-linked, [alpha](1->6)-branched glucans whose shape, cellular location, and structure differ markedly from those of storage starch. That synthesis of these two types of polysaccharides is controlled by both a common and a specific set of genes was evidenced by the identification of a new Chlamydomonas (STA4) locus specifically involved in the biosynthesis of storage starch. Mutants defective in STA4 accumulated a new type of high-amylose storage starch displaying an altered amylopectin chain size distribution. It is expected that the dual nature and functions of starch synthesis in unicellular green algae will yield new insights into the biological reasons for the emergence of starch in the eukaryotic plant cell.

Toward an understanding of the biogenesis of the starch granule. Determination of granule-bound and soluble starch synthase functions in amylopectin synthesis.

Plant starch synthesis can be distinguished from those of bacterial, fungal, and animal glycogen by the presence of multiple elongation (starch synthases) and branching enzymes. This complexity has precluded genetic assignment of functions to the various soluble starch synthases in the building of amylopectin. In Chlamydomonas, we have recently shown that defects in the major soluble starch synthase lead to a specific decrease in the amount of a subset of amylopectin chains whose length ranges between 8 and 40 glucose residues (Fontaine, T., D'Hulst, C., Maddelein, M.-L., Routier, F., Marianne-Pepin, T., Decq, A., Wieruszeski, J. M., Delrue, B., Van Den Koornhuyse, N., Bossu, J.-P., Fournet, B., and Ball, S. G. (1993) J. Biol. Chem. 268, 16223-16230). We now demonstrate that granule-bound starch synthase, the enzyme that was thought to be solely responsible for amylose synthesis, is involved in amylopectin synthesis. Disruption of the Chlamydomonas granule-bound starch synthase structural gene establishes that synthesis of long chains by this enzyme can become an absolute requirement for amylopectin synthesis in particular mutant backgrounds. In the sole presence of soluble starch synthase I, Chlamydomonas directs the synthesis of a major water-soluble polysaccharide fraction and minute amounts of a new type of highly branched granular material, whose structure is intermediate between those of glycogen and amylopectin. These results lead us to propose that the nature of the elongation enzyme conditions the synthesis of distinct size classes of glucans in all starch fractions.