Studies on Molecular Mechanism of Starch Biosynthesis

RBE 1、RBE 3和RBE 4的cDNA克隆是从一个发育中的水稻种子cDNA文库中鉴定的。RBE 1、RBE 3和RBE 4的成熟蛋白分别含有756、760和788个氨基酸，计算分子量分别为86，734、86，376和89，745 Da。RBE 1分别与RBE 3和RBE 4具有显著程度的序列同一性（49%和47%），而RBE 3序列与RBE 4的序列具有80%的同一性。分支酶的这些同种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，形成淀粉分解酶催化位点的四个区域的共有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酶属于淀粉分解酶家族。这一事实表明，分支酶具有两种酶功能，如α-1，4-糖苷键的断裂和新形成的还原酶的转移。 ...更多信息 g末端连接到其它α-1，4-连接的链。水稻分支酶的三种异构体之间存在两个结构差异：RBE 1在羧基端具有约50个残基的额外序列，并且与RBE 3和RBE 4相比，缺少近70个残基的氨基端序列（RBE 4为90个残基）。另一个区别是RBE 1序列含有一个8-残基序列，该序列特异性地存在于该同种型的羧基末端一半。RBE 3和RBE 4具有RBE 1缺失的11个残基的特异性序列。为了研究水稻分支酶3种异构体的酶功能，我们建立了水稻分支酶3种异构体cDNA片段在大肠杆菌中的表达系统。我们还制备了许多突变蛋白，包括RBE 1和RBE 3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”，以及将半胱氨酸残基替换为丝氨酸的“点突变蛋白”。我们的数据表明，RBE 1具有比RBE 3和RBE 4高得多的支化直链淀粉和支链淀粉的比活性。一个有趣的观察是，RBE 4能够支化直链淀粉的速度几乎比RBE 3快3倍，尽管这两种异构体之间的序列相似性相当高。然而，这三种异构体从直链淀粉产生的支链葡聚糖的单元链分布相对相似。突变蛋白的分析表明，从氨基末端的第三，第六和第七个半胱氨酸残基是重要的RBE 1的活性。它也可以得出结论，羧基端和氨基端序列的约50和60个残基的RBE 1和RBE 4，分别是不必要的酶的活性。尽管如此，靠近两端的氨基酸序列的差异似乎仍然解释了三种异构体之间不同的酶功能。少

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