DPH5, A METHYLTRANSFERASE GENE REQUIRED FOR DIPHTHAMIDE BIOSYNTHESIS IN SACCHAROMYCES-CEREVISIAE
DPH5, A METHYLTRANSFERASE GENE REQUIRED FOR DIPHTHAMIDE BIOSYNTHESIS IN SACCHAROMYCES-CEREVISIAE
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DOI：
10.1128/mcb.12.9.4026
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发表时间：
1992-09-01
影响因子：
5.3
通讯作者：
COLLIER, RJ
中科院分区：
文献类型：
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作者：
MATTHEAKIS, LC;SHEN, WH;COLLIER, RJ
通过白喉毒素（DT）的F2片段的细胞内表达选择了在S-腺苷甲硫氨酸（ADMet）依赖的甲基转移酶步骤中有缺陷的酿酒酵母的突变体，并显示其属于互补组DPH 5。对DPH 5基因进行克隆、测序，发现其编码一种300个残基的蛋白质，该蛋白质与细菌的尿卟啉原III甲基转移酶（uropyrinogen III methyltransferases）具有序列相似性，尿卟啉原III甲基转移酶是参与钴胺素（维生素B-12）生物合成的酶。这两个DPH 5和DPH 5：尿卟啉原III甲基转移酶缺乏序列基序常见于其他甲基转移酶，并可能代表一个新的DPH 5：甲基转移酶家族。DPH 5蛋白在大肠杆菌中产生，并显示在从dph 5突变体部分纯化的延伸因子2的甲基化中具有活性。染色体DPH 5基因的无效突变不影响细胞活力，与其他研究一致，表明细胞存活不需要二苯二甲酰胺。dph 5无效突变体在三种酶减毒DT片段的表达下存活，但被完全活性DT片段A的表达杀死。与这些结果一致，发现来自dph 5无效突变体的延伸因子2具有弱的ADP-核糖基受体活性，其仅在高浓度片段A存在下可检测到。
A mutant of SaccharomyceN cerevisiae defective in the S-adenosylmethionine (AdoMet)-dependent methyl-transferase step of diphthamide biosynthesis was selected by intracellular expression of the F2 fragment of diphtheria toxin (DT) and shown to belong to complementation group DPH5. The DPH5 gene was cloned, sequenced, and found to encode a 300-residue protein with sequence similarity to bacterial AdoMet: uroporphyrinogen III methyltransferases, enzymes involved in cobalamin (vitamin B-12) biosynthesis. Both DPH5 and AdoMet:uroporphyrinogen III methyltransferases lack sequence motifs commonly found in other methyltransferases and may represent a new family of AdoMet:methyltransferases. The DPH5 protein was produced in Escherichia coli and shown to be active in methylation of elongation factor 2 partially purified from the dph5 mutant. A null mutation of the chromosomal DPH5 gene did not affect cell viability, in agreement with other studies indicating that diphthamide is not required for cell survival. The dph5 null mutant survived expression of three enzymically attenuated DT fragments but was killed by expression of fully active DT fragment A. Consistent with these results, elongation factor 2 from the dph5 null mutant was found to have weak ADP-ribosyl acceptor activity, which was detectable only in the presence of high concentrations of fragment A.