Adenylate kinases from thermosensitive Escherichia coli strains.
The adk genes from several thermosensitive (ts) mutants of Escherichia coli were cloned and sequenced. The mutations responsible for the thermolability of the gene product, the enzyme adenylate kinase, were established. From five independently isolated strains analysed, two contain a CCG to TCG transition changing proline 87 to serine (P87S), another two have a TCT to TTT transition that mutates serine 129 to phenylalanine (S129F), and the last one was found not to contain a mutation in the adk gene. Overproducing strains were constructed that contain ts genes in the genome as well as in the plasmids. These strains grow at high temperature, although much slower than wild-type. Most probably, the high rate of synthesis of adenylate kinase compensates for the destruction of the thermolabile protein by the elevated temperature. Mutated proteins were purified. The P87S but not the S129F mutation was found to cause thermosensitivity of the adenylate kinase reaction. Revertants of thermosensitivity were isolated and the nature of the mutation was determined by the RNase digestion method of RNA-DNA hybrids and by DNA sequencing. The revertants of the P87S mutation regained the wild-type sequence, whereas the revertants of the S129F strain retained the original mutation in the adenylate kinase gene. These results are discussed in the light of the three-dimensional structure of the enzyme and the possible role of adenylate kinase in phospholipid synthesis.
Haase
G H
GH
Abteilung Biophysik, Max-Planck-Institut für Medizinische Forschung, Heidelberg, FRG.
Brune
M
M
Reinstein
J
J
Pai
E F
EF
Pingoud
A
A
Wittinghofer
A
A
D000263:Adenylate Kinase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009154:Mutation; D010770:Phosphotransferases / Q000235:genetics*; D012260:Ribonucleases / Q000378:metabolism
D001426:Bacterial Proteins; D010770:Phosphotransferases; D000263:Adenylate Kinase; D012260:Ribonucleases