No abstract

SummaryDisproportionating enzyme (D-enzyme) is a plastidial a-1,4-glucanotransferase but its role in starch metabolism is unclear. Using a reverse genetics approach we have isolated a mutant of Arabidopsis thaliana in which the gene encoding this enzyme (DPE1) is disrupted by a T-DNA insertion. While Denzyme activity is eliminated in the homozygous dpe1±1 mutant, changes in activities of other enzymes of starch metabolism are relatively small. During the diurnal cycle, the amount of leaf starch is higher in dpe1±1 than in wild type and the amylose to amylopectin ratio is increased, but amylopectin structure is unaltered. The amounts of starch synthesised and degraded are lower in dpe1±1 than in wild type. However, the lower amount of starch synthesised and the higher proportion of amylose are both eliminated when plants are completely de-starched by a period of prolonged darkness prior to the light period. During starch degradation, a large accumulation of malto-oligosaccharides occurs in dpe1±1 but not in wild type. These data show that D-enzyme is required for malto-oligosaccharide metabolism during starch degradation. The slower rate of starch degradation in dpe1±1 suggests that maltooligosaccharides affect an enzyme that attacks the starch granule, or that D-enzyme itself can act directly on starch. The effects on starch synthesis and composition in dpe1±1 under normal diurnal conditions are probably a consequence of metabolism at the start of the light period, of the high levels of maltooligosaccharides generated during the dark period. We conclude that the primary function of D-enzyme is in starch degradation.

The amylose fraction of starch occurs in double-helical A-and B-amyloses and the single-helical V-amylose. The latter contains a channel-like central cavity that is able to include molecules, ''iodine's blue'' being the best-known representative. Molecular models of these amylose forms have been deduced by solid state 13 C cross-polarization͞magic angle spinning NMR and by x-ray fiber and electron diffraction combined with computer-aided modeling. They remain uncertain, however, as no structure at atomic resolution is available. We report here the crystal structure of a hydrated cycloamylose containing 26 glucose residues (cyclomaltohexaicosaose, CA26), which has been determined by real͞reciprocal space recycling starting from randomly positioned atoms or from an oriented diglucose fragment. This structure provides conclusive evidence for the structure of V-amylose, as the macrocycle of CA26 is folded into two short left-handed V-amylose helices in antiparallel arrangement and related by twofold rotational pseudosymmetry. In the V-helices, all glucose residues are in syn orientation, forming systematic interglucose O(3) n ⅐⅐⅐O(2) n؉l and O(6) n ⅐⅐⅐O (2) Starch is composed of two fractions, the linear amylose consisting exclusively of ␣(1-4)-linked glucose residues in 4 C 1 -chair conformation, and the branched amylopectin, which also contains ␣(1-6) links at characteristic intervals. The polysaccharide chain of amylose may be folded into three different structures denoted A, B, and V (1-3). Since crystallization of amylose fragments with defined chain lengths has remained elusive, structural information relies on x-ray fiber diffraction, electron diffraction on tiny single crystals, and solid-state 13 C cross-polarization͞magic angle spinning (CP͞MAS) NMR spectroscopy combined with computer-aided modeling (3-8). The only available single crystal x-ray study of an amylose-type oligosaccharide in the complex (p-nitrophenyl ␣-maltohexaoside) 2 ⅐Ba(I 3 ) 2 ⅐27H 2 O features an antiparallel left-handed double helix (9, 10) that has no resemblance to A-, B-, or V-amylose.The structures of A-and B-amylose are similar and differ only in packing arrangement and water content, the A-form occurring preferentially in cereals and the B-form in tubers (3). They both form double helices with parallel strands of 6 ϫ 2 glucoses per turn and right-handed (3-8) or left-handed (11) twist, this ambiguity illustrating the weakness of the above methods, which do not provide structural information at atomic resolution.The polysaccharide chain of V-amylose found naturally in non-A and non-B segments of amylose is folded into a lefthanded single helix; it contains 6 glucoses per turn with 7.91-to 8.17-Å pitch height (3-5) and forms a central channel-like cavity.