Chen Xiaona, Shen Dan, Zhou Bing
State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China.
Biochem Biophys Res Commun. 2006 Jun 30;345(2):834-42. doi: 10.1016/j.bbrc.2006.04.101. Epub 2006 May 2.
Pantothenate kinase (PanK), a key regulatory enzyme in the coenzyme A (CoA) biosynthetic pathway, catalyzes the rate-limiting phosphorylation of pantothenic acid to form phosphopantothenate during CoA biosynthesis. Escherichia coli ts9 strain manifests temperature-sensitive phenotype on LB media due to its mutation in the coaA gene (coaA1). Sequencing analysis revealed that coaA1 arises from a single base pair mutation that results in an amino acid change, L236F. This change, located proximate to the ATP binding site of CoaA, destabilizes both enzymatic activity and structural integrity or stability of the mutant protein in vitro. Spontaneously, revertants of ts9 were occasionally found on LB medium plates. Two groups of revertants were isolated: for those that can grow at 40 degrees C, a reversion of the original amino acid mutation L236F to L236L or other amino acid (such as L236C) occurs; for those that can grow at 37 degrees C but not 40 degrees C, a mutation at another gene or intergenic suppression is strongly indicated. Towards genetic identification of genes that might interact with coaA1, ybjN, which encodes a putative sensory transduction regulator protein, and whose over-expression is capable of ameliorating the temperature-sensitive phenotype of the structurally unstable CoaA1 or CoaA[L236F], was isolated. Over-expression of ybjN appears to suppress the temperature-sensitive phenotype of several other temperature-sensitive mutations, including coaA14 (carried by DV51 strain), coaA15 (carried by DV70 strain), and ilu-1, suggesting it not only helps CoaA1, but possibly works as a general stabilizer for some other unstable proteins.
泛酸激酶（PanK）是辅酶A（CoA）生物合成途径中的关键调节酶，在CoA生物合成过程中催化泛酸的限速磷酸化反应，生成磷酸泛酰巯基乙胺。大肠杆菌ts9菌株由于其coaA基因（coaA1）发生突变，在LB培养基上表现出温度敏感型表型。测序分析表明，coaA1源于单个碱基对突变，导致氨基酸变化，即L236F。这种变化位于CoaA的ATP结合位点附近，在体外使突变蛋白的酶活性以及结构完整性或稳定性均不稳定。偶尔在LB培养基平板上会自发出现ts9的回复突变体。分离出了两组回复突变体：对于那些能在40℃生长的回复突变体，原始氨基酸突变L236F回复为L236L或其他氨基酸（如L236C）；对于那些能在37℃生长但不能在40℃生长的回复突变体，则强烈表明是另一个基因发生了突变或存在基因间抑制。为了从基因层面鉴定可能与coaA1相互作用的基因，分离出了ybjN，它编码一种假定的感觉转导调节蛋白，其过表达能够改善结构不稳定的CoaA1或CoaA[L236F]的温度敏感型表型。ybjN的过表达似乎能抑制其他几种温度敏感型突变的温度敏感型表型，包括coaA14（由DV51菌株携带）、coaA15（由DV70菌株携带）和ilu - 1，这表明它不仅对CoA1有帮助，还可能作为一些其他不稳定蛋白的通用稳定剂发挥作用。
