Starch debranching enzyme (R-enzyme or pullulanase) from developing rice endosperm: purification, cDNA and chromosomal localization of the gene

Abstract

Starch debranching enzyme (R-enzyme or pullulanase) was purified to homogeneity from developing endosperm of rice (Oryza sativa L. cv. Fujihikari) using a variety of high-performance liquid chromatography columns, and characterized. A cDNA clone encoding the full length of the rice endosperm debranching enzyme was isolated and its nucleotide sequence was determined. The cDNA contains an open reading frame of 2958 bp. The mature debranching enzyme of rice appears to be composed of 912 amino acids with a predicted relative molecular mass (Mr) of 102,069 Da, similar in size to its Mr of about 100,000 Da estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The amino acid sequence of rice debranching enzyme is substantially similar to that of bacterial pullulanase, while it bears little similarity to that of bacterial isoamylase or to glycogen debranching enzymes from human muscle and rabbit muscle. Southern blot analyses strongly suggest that the debranching enzyme gene is present as a single copy in the rice genome. Analysis by restriction fragment length polymorphism with a probe including the 3'-untranslated region of cDNA for rice debranching enzyme confirmed that the debranching enzyme gene is located on chromosome 4.

Effect of High Temperature at Ripening Stage on the Reserve Accumulation in Seed in Some Rice Cultivars

“…such as insensitive response of starch synthase in rice endosperm to high temperature compared with wheat endosperm (Rijven, 1986) or possible role of debranching enzymes in starch degradation (Nakamura et al, 1996) have been reported, ADP -glucose pyrophosphorylase and soluble starch synthase seem to be one of key enzymes under high-temperature stress. Further studies on the behavior of these enzymes are needed.…”

A Single Limit Dextrinase Gene Is Expressed Both in the Developing Endosperm and in Germinated Grains of Barley1

“…For this reason, and because there is a marked preference for the hydrolysis of (136)-␣-glucosyl linkages in oligosaccharides compared with polysaccharides, the enzyme is usually referred to as limit dextrinase (Lee et al, 1971). This nomenclature will be used here, although the enzyme is also referred to as the R-enzyme (Nakamura et al, 1996).As a result of the proposed role for limit dextrinases in starch hydrolysis in germinated barley grains, and because of the associated importance of this process in the malting and brewing industries, considerable effort has been directed toward the purification and characterization of the barley enzyme (Sissons et al, 1992;Longstaff and Bryce, 1993;MacGregor et al, 1994a;Kristensen et al, 1998). The enzyme has been reported in both developing (Sissons et al, 1993) and germinated grain (Kristensen et al, 1998).…”

“…For this reason, and because there is a marked preference for the hydrolysis of (136)-␣-glucosyl linkages in oligosaccharides compared with polysaccharides, the enzyme is usually referred to as limit dextrinase (Lee et al, 1971). This nomenclature will be used here, although the enzyme is also referred to as the R-enzyme (Nakamura et al, 1996).…”

“…A debranching enzyme would also be capable of producing primers for the starch synthase reaction if required (Duffus and Cochrane, 1993), a function that would explain the presence of limit dextrinase in developing rice grain (Nakamura et al, 1996).…”

Recent progress toward understanding the role of starch biosynthetic enzymes in the cereal endosperm

“…The function of PUL is less understood than that of ISA. PUL is believed to be associated with the degradation of starch during kernel germination; however, its activity has been detected in developing rice and maize endosperms [106][107][108], suggesting PUL may also be involved in starch biosynthesis. Loss of just PUL in maize mutants results in no significant alterations in starch structure or composition in maize endosperm, while loss of both ISA1 and PUL results in a significant accumulation of phytoglycogen in maize endosperm [109].…”