Genes of the thymidine salvage pathway:: Thymine-7-hydroxylase from a Rhodotorula glutinis cDNA library and iso-orotate decarboxylase from Neurospora crassa
Genes of the thymidine salvage pathway:: Thymine-7-hydroxylase from a Rhodotorula glutinis cDNA library and iso-orotate decarboxylase from Neurospora crassa
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DOI：
10.1016/j.bbagen.2005.02.001
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发表时间：
2005-05-25
影响因子：
3
通讯作者：
Axhemi, AA
中科院分区：
文献类型：
--
作者：
Smiley, JA;Kundracik, M;Axhemi, AA
胸苷补救途径中的两种酶的基因，胸腺嘧啶-7-羟化酶（THase;正式名称胸腺嘧啶双加氧酶）和异乳清酸脱羧酶（IDCase）已经从真菌来源中分离。基于已发表的氨基酸序列，使用简并寡核苷酸从红酵母cDNA文库中分离THase。将编码序列转移到大肠杆菌表达系统中，使用C-14标记的胸腺嘧啶测量重组THase活性。THase序列显示几乎完全避免以A或T结尾的密码子：95.8%的GC含量存在于密码子的第三位置。这种密码子的偏见和嘧啶代谢中的胸苷补救途径的作用之间的连接提出。THase序列类似于I组Fe+2依赖性、α KG依赖性双加氧酶。中华民国利用已测序的粗糙脉孢菌和构巢曲霉的THase基因，对可能存在的THase基因进行了定位。这两个基因组中与THase相邻的基因彼此相似，并且与哺乳动物2-氨基-3-羧基粘康酸-6-半醛脱羧酶（ACMSD）相似，导致它们被鉴定为IDCase基因。了N.通过基因组DNA的PCR分离crassa版本，并在重组E.携带该基因的大肠杆菌。一个新的家庭的脱羧酶，使用类似的底物，确定凭借的蛋白质序列的相似性。(c)2005 Elsevier B. V.保留所有权利。
Genes for two enzymes in the thymidine salvage pathway, thymine-7-hydroxylase (THase; official name thymine dioxygenase) and isoorotate decarboxylase (IDCase) have been isolated from fungal sources. THase was isolated from a Rhodotorula glutinis cDNA library using a degenerate oligonucleotide based on the published amino acid sequence. The coding sequence was transferred to an Escherichia coli expression system, from which recombinant THase activity was measured using C-14-labeled thymine. The THase sequence shows an almost complete avoidance of codons ending in A or T: 95.8% GC content is present in the third position of codons. A connection between this codon bias and the role of the thymidine salvage pathway in pyrimidine metabolism is proposed. The THase sequence is similar to Group I Fe+2-dependent, alpha KG-dependent dioxygenases. The R. glutinis THase gene was used to locate the probable THase genes in the sequenced genomes of Neurospora crassa and Aspergillus nidulans. The genes neighboring THase in these two genomes are similar to each other, and are similar to the mammalian 2-amino-3-carboxymuconate-6-semialdhyde decarboxylase (ACMSD), leading to their identification as IDCase genes. The N. crassa version was isolated by PCR of genomic DNA, and IDCase activity was measured in recombinant E. coli carrying this gene. A new family of decarboxylases, using similar substrates, is identified by virtue of the protein sequence similarity. (c) 2005 Elsevier B.V. All rights reserved.