Studies on Molecular Mechanism of Starch Biosynthesis

淀粉生物合成的分子机制研究

• 批准号: 07660092
• 负责人: BABA Tadashi
• 金额: $ 1.47万
• 依托单位: University of Tsukuba
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07660092/
• 关键词: Starch Synthesis; Amylose; Amylopectin; Branching Enzyme; Starch Synthase; Rice; Gene Cloning; Recombinant Enzyme; アシロース; 板つけ酵素; 澱粉生合成

The cDNA clones coding for RBE1, RBE3, and RBE4 have been identified from a developing rice seed cDNA library in lambdagt11. The mature proteins of RBE1, RBE3, and RBE4 contain 756,760, and 788 amino acids with calculated molecular masses of 86,734,86,376, and 89,745 Da, respectively. RBE1 shares a noticeable degree of sequence identity (49 and 47%) with RBE3 and RBE4, respectively, whereas the RBE3 sequence is 80% identical to that of RBE4. These isoforms of braching enzyme all share a high degree of sequence identity in the middle regions of the protein molecules. Interestingly, the consensus sequences of the four regions, which form the catalytic sites of amylolytic enzymes, are conserved in the regions. Thus, plant branching enzyme as well as the bacterial and mammalian enzymes belongs to a family of amylolytic enzymes. This fact implies that branching enzyme possesses two enzymatic functions such as cleavage of alpha-1,4-glucosidic linkages and transfer of the newly formed reducin … More g ends to other alpha-1,4-linked chains. There are two structural differences among three isoforms of rice branching enzyme ; RBE1 possesses an approximately 50-residue extra sequence at the carboxyl terminus, and lacks an amino-terminal sequence of almost 70 residues (90 residues for RBE4), as compared with RBE3 and RBE4. Another difference is that the RBE1 sequence contains an 8-residue sequence specifically present in this isoform at the carboxyl-terminal half. On the contrary, RBE3 and RBE4 have an 11-residue specific sequence that is deficient in RBE1.To examine enzymatic functions of three isoforms of rice branching enzyme, we have established an expression system of the cDNA fragments in Escherichia coli. We have also prepared a lot of mutant proteins, including chimeric enzymes between RBE1 and RBE3, "deletion proteins" without the amino-or carboxyl-terminal sequences of the mature enzymes, and "point-mutated proteins" with the replacement of cysteine residue (s) into serine. Our data demonstrate that RBE1 has the much higher specific activity in branching amylose and amylopectin than RBE3 and RBE4. An intriguing observation is that RBE4 is capable of branching amylose almost 3-fold more rapidly than RBE3 in spite of the considerably high degree of sequence similarity between these two isoforms. However, the unit-chain profiles of branched glucans produced from amylose by these three isoforms are relatively similar. Analysis of the mutant proteins indicates that third, sixth, and seventh cysteine residues from the amino terminus are important for the activity of RBE1. It is also concluded that the carboxyl-terminal and amino-terminal sequences of approximately 50 and 60 residues in RBE1 and RBE4, respectively, are not essential for the enzyme activity. Despite this fact, the difference of the amino acid sequences near both termini still seems to explain the distinct enzymatic functions among three isoforms. Less

从一个发育中的水稻种子cDNA文库中鉴定出了RBE1、RBE3和RBE4的cDNA克隆。RBE1、RBE3和RBE4的成熟蛋白分别含有756,760和788个氨基酸，计算出的分子质量分别为86,734、86,376和89,745 Da。RBE1与RBE3和RBE4的序列一致性显著（分别为49%和47%），而RBE3与RBE4的序列一致性为80%。这些支链酶的同工型在蛋白质分子的中间区域都具有高度的序列同一性。有趣的是，形成淀粉解酶催化位点的四个区域的一致序列在这些区域中是保守的。因此，植物分支酶以及细菌和哺乳动物的酶都属于一个解淀粉酶家族。这一事实表明分支酶具有两种酶促功能，即α -1,4-糖苷键的裂解和新形成的还原蛋白的转移。水稻分枝酶的三种同工异构体存在两种结构差异；与RBE3和RBE4相比，RBE1在羧基端有大约50个残基的额外序列，缺少近70个残基的氨基端序列（RBE4有90个残基）。另一个不同之处在于，RBE1序列包含一个8个残基序列，专门存在于该同工异构体的羧基末端。相反，RBE3和RBE4具有RBE1缺失的11个残基特异性序列。为了研究水稻分枝酶三种同工异构体的酶功能，我们建立了一个cDNA片段在大肠杆菌中的表达系统。我们还制备了大量的突变蛋白，包括RBE1和RBE3之间的嵌合酶，没有成熟酶的氨基或羧基末端序列的“缺失蛋白”，以及半胱氨酸残基被丝氨酸取代的“点突变蛋白”。我们的数据表明，RBE1在分支直链淀粉和支链淀粉方面比RBE3和RBE4具有更高的特异性活性。一个有趣的观察是，RBE4能够分支直链淀粉比RBE3快近3倍，尽管这两个亚型之间的序列相似性相当高。然而，这三种同工型由直链淀粉产生的支链葡聚糖的单位链谱是相对相似的。突变蛋白的分析表明，氨基末端的第3、第6和第7个半胱氨酸残基对RBE1的活性很重要。RBE1和RBE4中分别有大约50个和60个残基的羧基端和氨基端序列对酶活性不是必需的。尽管如此，两个末端附近氨基酸序列的差异似乎仍然可以解释三种同工异构体之间不同的酶功能。少

项目成果

Baba, T.: "Studies on Molecular Mechanism of Starch Biosynthesis" Nippon Nogeikagaku Kaishi. 71. 129-136 (1997)

Baba, T.：“淀粉生物合成的分子机制研究”Nippon Nogeikagaku Kaishi。

Ken-ichiroh tanakaら: "Structure, organization, and chromosomal location of the gene encoding a form of rice soluble starch synthase" Plant Physiology. 108. 677-683 (1995)

Ken-ichiroh Tanaka 等：“编码一种稻可溶性淀粉合酶的基因的结构、组织和染色体位置”植物生理学 108. 677-683 (1995)。

Hiromori Akagi: "Nucleotide Sequence of a Stearoyl-Acyl Carrier Protein Desaturase from Developing Seeds of Rice" Plant Physiology. 108. 845-846 (1995)

Hiromori Akagi：“来自发育中的水稻种子的硬脂酰酰基载体蛋白去饱和酶的核苷酸序列”植物生理学。

Tsutomu Kawasaki: "Coordinated Regulation of the Gene Participating in Starch Biosynthesis by the Rice Floury-2 Locus" Plant Physiology. 110. 89-96 (1996)

Tsutomu Kawasaki：“水稻Floury-2位点参与淀粉生物合成的基因的协调调节”植物生理学。

Tsutomu Kawasakiら: "Coordinated regulation of the genes participating in starch biosynthesis by the rice Floury-2 locus" Plant Physiology. 110. 89-96 (1996)

Tsutomu Kawasaki 等：“水稻 Flory-2 基因座参与淀粉生物合成的基因的协调调节”植物生理学 110. 89-96 (1996)。