Analysis of the temperature-sensitive mutation of Escherichia coli pantothenate kinase reveals YbjN as a possible protein stabilizer.
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.
Chen
Xiaona
X
State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China.
Shen
Dan
D
Zhou
Bing
B
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D003065:Coenzyme A / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D009154:Mutation; D010641:Phenotype; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013696:Temperature
D029968:Escherichia coli Proteins; C512428:YbjN protein, E coli; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C023088:pantothenate kinase; D003065:Coenzyme A