Secretion of mono- and diacylglycerol lipase from Penicillium camembertii U-150 by Saccharomyces cerevisiae and site-directed mutagenesis of the putative catalytic sites of the lipase.
Yeast cells carrying intronless mono- and diacylglycerol lipase (MDGL) genes, constructed by recombination of the genomic gene and cDNA, secreted MDGL into the culture supernatant. Most of the yeast MDGL were extensively glycosylated while they had a similar glyceride specificity to that of native MDGL. Site-directed mutagenesis was used to directly confirm the involvements in enzyme activity of the presumptive amino acid residues to form the catalytic center of MDGL. These residues were conserved in the primary structure alignment of a lipase family from filamentous fungi. Mutant lipase proteins in which Ser83, Ser145, or His259 was replaced with glycine were secreted by yeast transformants as inactive proteins. Mutant proteins replacing Asp199 with glycine or asparagine were not detected in the culture supernatant. Replacing other two highly conserved aspartic acids (at positions 232 and 243) with glycine did not render the enzyme inactive. These results indicate that Ser83, Ser145, and His259 in MDGL, are essential to enzyme activity. Asp199 is also likely to be involved.
Yamaguchi
S
S
Tsukuba Research Laboratories, Amano Pharmaceutical Co., Ltd., Ibaraki, Japan.
Mase
T
T
Takeuchi
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004274:DNA, Recombinant / Q000737:chemistry; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics; D008071:Lipoprotein Lipase / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D008994:Monoacylglycerol Lipases / Q000096:biosynthesis* / Q000235:genetics; D016296:Mutagenesis; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D008994:Monoacylglycerol Lipases; D008049:Lipase; D008071:Lipoprotein Lipase