Cloning, expression, and characterization of a thermostable PAP2L2, a new member of the type-2 phosphatidic acid phosphatase family from Geobacillus toebii T-85

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2008-12, Vol.72 (12), p.3134-3141
• Main Authors: Zhang, Y.(Fudan Univ., Shanghai (China)), Yang, Z, Huang, X, Peng, J, Fei, X, Gu, S, Xie, Y, Ji, C, Mao, Y
• Format: Article
• Language: English
• Subjects: ACID PHOSPHATASE; Amino Acid Sequence; ANIMAL; ANIMALES; ANIMALS; Bacillaceae - enzymology; Base Sequence; Biological and medical sciences; CLONACION; CLONAGE; CLONING; Cloning, Molecular; Enzyme Stability; Escherichia coli; Escherichia coli - genetics; EXPRESION GENICA; EXPRESSION DES GENES; FOSFATASA ACIDA; FOSFOLIPIDOS; Fundamental and applied biological sciences. Psychology; GENE EXPRESSION; Geobacillus toebii; HEAT TOLERANCE; integral membrane phosphatase; Molecular Sequence Data; PHOSPHATASE ACIDE; Phosphatidate Phosphatase - chemistry; Phosphatidate Phosphatase - genetics; Phosphatidate Phosphatase - isolation & purification; Phosphatidate Phosphatase - metabolism; PHOSPHATIDE; PHOSPHOLIPIDS; PROTEINAS; PROTEINE; PROTEINS; PURIFICACION; PURIFICATION; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Analysis, DNA; Temperature; thermostability; TOLERANCE A LA CHALEUR; TOLERANCIA AL CALOR; type 2 phosphatidic acid phosphatase
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.80305

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 Nisup(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 60degC. The enzyme was strongly inhibited by 1% SDS, 10 mM veranda, and Znsup(2+), whereas it was independent of the Mgsup(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.