Cloning, expression, and characterization of a thermostable PAP2L2, a new member of the type-2 phosphatidic acid phosphatase family from Geobacillus toebii T-85.
Most members of the type-2 phosphatidic acid phosphatase (PAP2) superfamily are integral membrane phophatases involved in lipid-related signal transduction and metabolism. Here we describe the cloning of a novel gene from Geobacillus toebii T-85, encoding a PAP2-like protein, Gtb PAP2L2, which contains 212 amino acids and shows a limited homology to other known PAP2s, especially at conserved phosphatase motifs, and a similar six-transmembrane topology structure. This enzyme was expressed, and purified in Escherichia coli. Recombinant Gtb PAP2L2s from the membrane fractions were solublized with 0.3% (v/v) Triton X-100 and purified by Ni(2+) affinity chromatography. The purified enzyme showed broad substrate specificity to phosphatidic acid, diacylglycerol pyrophosphate, and lysophosphatidic, but preferred phosphatidic acid and diacylglycerol pyrophosphate in vitro. Gtb PAP2L2 is a thermal stable enzyme with a half-life of 30 min at 60 degrees C. The enzyme was strongly inhibited by 1% SDS, 10 mM veranda, and Zn(2+), whereas it was independent of the Mg(2+) ion, and insensitive to N-ethylmaleimide. The purified recombinant Gtb PAP2L2 was catalytically active and highly stable, making it ideal as a candidate on which to base further PAP2 structure/function studies.
Zhang
Yong
Y
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, PR China.
Yang
Zhenxing
Z
Huang
Xiaodong
X
Peng
Jing
J
Fei
Xiangwei
X
Gu
Shaohua
S
Xie
Yi
Y
Ji
Chaoneng
C
Mao
Yumin
Y
D000595:Amino Acid Sequence; D001406:Bacillaceae / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D010711:Phosphatidate Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature
D011994:Recombinant Proteins; C406242:phosphatidic acid phosphatase type 2; D010711:Phosphatidate Phosphatase