Nucleotide sequence of a novel arylesterase gene from Vibro mimicus and characterization of the enzyme expressed in Escherichia coli.
A gene coding for an arylesterase of Vibrio mimicus was cloned. Sequence determination reveals that the esterase gene has an open reading frame of 600 nucleotides which encodes a protein of M(r) 22,300. The deduced amino acid sequence contain a pentapeptide GDSLS (residues 27-31), which was also found in the phospholipid-cholesterol acyltransferase from Aeromonas hydrophila. Substitution of Ser-29 by alanine or cysteine in the cloned gene abolished the esterase activity in the tributyrin plate assay. On the other hand, the activity was not lost when Ser-31 was changed to alanine. The cloned gene was expressed in Escherichia coli, and the protein purified by a four-step procedure. The purified protein migrated on SDS/PAGE as a single band with an apparent M(r) of 22,100. This enzyme favoured the hydrolysis of several arylesters and was classified as an arylesterase (EC 3.1.1.2). N-Terminal analysis showed that Ser-20 was the first amino acid of the mature secreted protein, suggesting that the N-terminal 19 hydrophobic amino acids served as a signal peptide.
Shaw
J F
JF
Institute of Botany, Academia Sinica, Taipei, Taiwan, Republic of China.
Chang
R C
RC
Chuang
K H
KH
Yen
Y T
YT
Wang
Y J
YJ
Wang
F G
FG
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012694:Serine; D014733:Vibrio / Q000201:enzymology / Q000235:genetics*
D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012694:Serine; D002265:Carboxylic Ester Hydrolases; C019734:arylesterase