Cloning, sequencing, and characterization of Escherichia coli thioesterase II.
The gene (tesB) encoding Escherichia coli thioesterase II, a low-abundance enzyme of unknown physiological function which can hydrolyze a broad range of acyl-CoA thioesters, has been localized by transposon mutagenesis, cloned and sequenced. A two-cistron construct containing both the lac and tesB promoters was used successfully to overexpress the 286-residue polypeptide. The recombinant enzyme constituted up to 25% of the soluble proteins of E. coli and was readily purified to homogeneity as a tetramer of approximately 120,000 Da. Amino-terminal sequence analysis and electrospray ionization mass spectrometry confirmed the identity of the thioesterase and revealed that the amino-terminal formyl-methionine had been removed yielding a subunit species of average molecular mass 31,842 Da. The protein does not contain the GXSXG motif found characteristically in animal thioesterases which function as chain-terminating enzymes in fatty acid synthesis and exhibits no sequence similarity with these or any other known proteins. Activity of the recombinant enzyme was inhibited by iodoacetamide and diethylpyrocarbonate. The carboxamidomethylated residue was identified as histidine 58, and a role for this amino acid in catalysis is suggested. E. coli strains having a large deletion within the genomic tesB gene grew normally but retained a low level of thioesterase activity toward decanoyl-CoA. This residual activity indicates the presence of an additional decanoyl-CoA hydrolase in E. coli. Over-expression of the recombinant enzyme, under control of the lac promoter, did not alter the fatty acids synthesized by E. coli at any stage of cell growth and the physiological role of this enzyme remains an enigma.
Naggert
J
J
Children's Hospital Oakland Research Institute, California 94609.
Narasimhan
M L
ML
DeVeaux
L
L
Cho
H
H
Randhawa
Z I
ZI
Cronan
J E
JE
Green
B N
BN
Smith
S
S
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003360:Cosmids; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D064429:Fatty Acid Synthases / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D010446:Peptide Fragments / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D013869:Thiolester Hydrolases / Q000235:genetics* / Q000378:metabolism
D004251:DNA Transposable Elements; D010446:Peptide Fragments; D012333:RNA, Messenger; D064429:Fatty Acid Synthases; D013869:Thiolester Hydrolases; C022738:thioesterase II