THE BIRA GENE OF ESCHERICHIA-COLI ENCODES A BIOTIN HOLOENZYME SYNTHETASE
THE BIRA GENE OF ESCHERICHIA-COLI ENCODES A BIOTIN HOLOENZYME SYNTHETASE
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DOI：
10.1016/0022-2836(81)90042-5
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发表时间：
1981-01-01
影响因子：
5.6
通讯作者：
CAMPBELL, AM
中科院分区：
文献类型：
--
作者：
BARKER, DF;CAMPBELL, AM
大肠杆菌biA基因突变导致生物素操纵子抑制、生物素摄取和细胞内生物素滞留的缺陷。该基因编码该生物体的主要生物素固定酶--乙酰辅酶A羧基酶生物素全酶合成酶(EC 6.3.4.15)。与野生型大肠杆菌提取物的情况不同，对超声提取物中蛋白质的标记生物素掺入的测量显示没有体外活性。三个不同的突变体表现出全酶合成酶活性的变化，包括一个由BirA361指定的不耐热活性的明显实例。用Lambda感染细胞扩增Bira基因表达携带大肠杆菌染色体EcoRI片段的噬菌体，包括该基因，导致比活性增加20-40倍。当lambda.BirA期携带BirA85突变时，没有观察到活性增加。用lambda.BirA361转导噬菌体感染细胞后，温度敏感活性增加了20-40倍。纯化了BirA361指定的活性.apprx。1000倍。纯化的酶比类似纯化的野生型酶更不耐热。提取液中全酶合成酶和整个细胞对生物素摄取的测定表明，某些突变位于与BIRA突变相同的染色体位置，但最初被认为是生物抑制缺陷，但生物素全酶合成酶和生物素摄取也缺乏。这一结果表明，该位置的所有突变都影响同一种酶，这些Bior突变被重新命名为BIRA。BIRA基因突变的互补性分析和该基因及其产物的生化鉴定结果支持BIRA产物具有生物阻遏和生物素全酶合成酶功能的观点。
Mutations in the birA gene of E. coli cause defects in biotin operon repression, biotin uptake and retention of intracellular biotin. The birA gene encodes the major biotin-fixing enzyme of this organism, the acetyl-CoA carboxylase biotin holoenzyme synthetase (EC 6.3.4.15). Unlike the situation in wild-type E. coli extracts, measurements of labeled biotin incorporation into protein in sonicated extracts reveal no in vitro activity. Three different mutants exhibit altered holoenzyme synthetase activity, including one clear instance of a thermolabile activity specified by birA361. Amplification of birA gene expression by infection of cells with a .lambda. phage bearing an EcoRI fragment of the E. coli chromosome which includes the gene results in a 20- to 40-fold increase in specific activity. When the .lambda.birA phase carries the birA85 mutation, no activity increase is observed. Infection of cells with a .lambda.birA361 transducing phage results in a 20- to 40-fold increase in temperature-sensitive activity. The activity specified by birA361 was purified .apprx. 1000-fold. The purified enzyme was more thermolabile than similarly purified wild-type enzyme. Measurements of holoenzyme synthetase in extracts and biotin uptake by whole cells indicate that certain mutations located at the same chromosomal position as birA mutations but initially characterized as defective only in bio repression are also deficient in biotin holoenzyme synthetase and biotin uptake. This result indicates that all mutations at this location affect the same enzyme and these bioR mutations were redesignated as birA. Results of complementation analysis of birA mutations and biochemical characterization of the gene and its product support the view that the birA product functions as the bio repressor and the biotin holoenzyme synthetase.