Abstract
The three-dimensional structure of lipase II of Geotrichum candidum strain ATCC34614 (GCL II) has provided insights with respect to the nature of the catalytic machinery of lipases. To support these structural observations, we have carried out an analysis of GCL II by mutagenesis. The gene encoding lipase II of Geotrichum candidum strain ATCC34614 (GCL II) was amplified using the polymerase chain reaction, cloned, and sequenced. The intronless lipase gene was expressed and secreted from Saccharomyces cerevisiae at approximately 5 mg/liter of culture. Recombinant GCL II was purified by immunoaffinity chromatography and characterized using a combination of substrates and independent analytical methods. The recombinant enzyme and the enzyme isolated from its natural source have comparable specific activities against triolein of about 1000 mumol of oleic acid released/min/mg of protein. The putative catalytic triad Ser217-His463-Glu354 was probed by site-directed mutagenesis. The substitution of Ser217 by either Cys or Thr and of His463 by Ala led to a complete elimination of the activity against both triolein and tributyrin. Substitution of Glu354 by either Ser, Ala or Gln renders the enzyme inactive and also perturbs the enzyme stability. However, the enzyme with the conservative replacement Glu354 Asp is stable and displays only a small decrease of triolein activity but a 10-fold decrease in activity against tributyrin. There was no appreciable difference in esterase activity between the native, recombinant wild type, and Glu354 Asp mutant. These results confirm that the triad formed by Ser217-Glu354-His463 is essential for catalytic activity. They also show that the active site of GCL II is more tolerant to a conservative change of the carboxylic side chain within the triad than are other hydrolases with similar catalytic triads.
Highlights
To support these struc- This activation was long ago proposed to be caused by a tural observations, we have carried out an analyosfis conformational changein the enzyme and GCL I1 by mutagenesis
Cloningand Sequencingof GCLZZ Gene from G. candidurnPrimers based on the GCL I1 cDNA sequence (Shimada et al, 1990) wereused to polymerase chain reaction amplify the complete corresponding gene from the genomic DNA of G. candidum ATCC 34614
The electron density of the refined 3-D structure of I1 were compared to natural GCL 11
Summary
Cloningand Sequencingof GCLZZ Gene from G. candidurnPrimers based on the GCL I1 cDNA sequence (Shimada et al, 1990) wereused to polymerase chain reaction amplify the complete corresponding gene from the genomic DNA of G. candidum ATCC 34614. Plasmid YpDC240 containing the GCL I1 gene fused to the a-factor prepro-region was assembled (Table I and Fig. 1).In this construction, secretion of GCL I1reliesupon efficientcleavage at the protein junction by the dibasic specific protease Kex (Julius et al, 1984). Sevendays after transfer intothe “optimum medium” (see “Experimental Procedures”) a maximum of 7.5 and 5.4 mg/liter of GCL I1was detected in the culture medium of BJ3501(YpDC240) and BJ3501(YpDC250), respectively This corresponds to less than 1%of the total protein present in the culture broth. No immunoreactive bands were detected in the culture medium of cthoentrol BJ3501(YpDC219), neither prior to (Fig. 3, lanes 7) nor following endoglycosidaseH treatment (Fig. 3, lanes 8).Most of the GCL I1 appears to be secreted, since only weak immunoreactive signals were detected in yeast cell extracts harboring YpDC240 (data notshown).
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