FEMS Microbiology Letters 100 (1992) 249-254 © 1992 Federation of European Microbiological Societies 0378-1097/92/$05.00 Published by Elsevier
Lipase of Staphylococcus hyicus: Analysis of the catalytic triad by site-directed mutagenesis Stephan J~iger, Gabriele Demleitner and Friedrich G6tz Mikrobielle Genetik, Universitiit Tiibingen, Tiibingen, FRG Received 9 June 1992 Accepted 30 July 1992
Key words: Lipase; Catalytic triad; Site-directed mutagenesis; Staphylococcus hyicus
1. SUMMARY In this study the putative catalytic triad SerHis-Asp of the Staphylococcus hyicus ssp. hyicus lipase was investigated. Putative catalytic sites determined by homology comparisons of three staphylococcal and other non-staphylococcal lipases were altered by site-directed mutagenesis. Since the mutations did not influence the secretion of the lipase, the decrease in lipase activity of the mutants strongly supports the proposed involvement of Ser 369 and His 6°° in catalysis. Asp 559 is postulated to be the third amino acid of the triad.
2. INTRODUCTION Lipases are non-specific esterases, typically activated by binding to lipid-water interfaces. Recently, the catalytic centres of five lipases were identified by X-ray crystallographic studies or
Correspondence to: F. G6tz, Universit~it Tiibingen, Auf der Morgenstelle 28, D-W7400 Tiibingen, FRG.
site-directed mutagenesis. The active centers of pancreatic lipase , human lipoprotein lipase , and the lipases of Rhizomucor miehei  and Fusarium solani  consist of the triad Ser-HisAsp and that of the Geotrichum candidum lipase  consists of Ser-His-Glu. In aqueous solution the catalytic center is hidden, but becomes accessible by binding to the lipid-water interface. All five known catalytic centers have an active serine which is part of the conserved pentapeptide GlyX1-Ser-X-Gly, with tyrosine or histidine in position X1. This conserved pentapeptide is also involved in the catalytic center of other esterases, i.e. thioesterases, esterases, acyltransferases and serine proteases [6, 7]. The catalytic triad SerHis-Asp of lipases is catalytically analogous to the Ser-His-Asp triad of serine proteases. So far, three staphylococcal lipases have been cloned and sequenced ([8,9]; Nikoleit, K. and G6tz, F., unpublished results), which share an identity of 64% in the mature protein . Because of the instability of these enzymes, possibly caused by autocatalytic serine protease activity or nonspecific degradation, X-ray crystallographic structures have not been determined. Therefore, the only current means of obtaining information
S. aureus U500
S. aureus PS54
Fig. l. Organization of the staphylococcal lipases as pre-proenzymes. SP, signal peptide; PP, propeptide.
about the catalytically active amino acids of these lipases is by chemical modification or site-directed mutagenesis at the putative active sites. The exolipase of Staphylococcus hyicus ssp. hyicus has been cloned in Staphylococcus carnosus TM300 on plasmid pLipPS1 [8,11-13]. The lipase is organized as a preprotein of 641 amino acids with a signal peptide of 38 amino acids. Although the molecular mass predicted from the DNA-sequence of the prolipase is 66.3 kDa, the apparent molecular mass from SDS-PAGE is approximately 86 kDa in S. carnosus and 46 kDa in S. hyicus, formed by cleavage of the propeptide between Thr 245 and Va1246 by a specific protease (Fig. 1) [13,14]. A remarkable property of the S. hyicus lipase is its broad substrate specificity. The enzyme hydrolyses not only lipids, but also phospholipids and lysophospholipids . The putative catalytically active serine, part of the conserved pentapeptide Gly-X1-Ser-X-Gly, is in position 369. His 269 has been proposed as the active histidine . In this work we show, by site-directed mutagenesis, that His 6°° and not His 269, and that Asp 559 and S e r 369 very likely comprise the catalytic triad of the S. hyicus lipase. Furthermore, we present evidence that the instability of the lipase is not due to intrinsic serine protease activity.
3. MATERIALS AND METHODS
3.1. Bacterial strains, plasmids and media Staphylococcus carnosus TM300  was used as the cloning host for lipase gene expression. Cells were cultivated in B-Medium (10 g of pep-
tone, 5 g of yeast extract, 5 g of NaC1, 1 g of glucose and 1 g of K2HPO 4 per liter). Cultures were grown overnight in the presence of 10 (mg chloramphenicol) 1 1. After cultivation, cells were removed by centrifugation, pLipPSS1, a derivative of pLipPS1 [13,14], contains an improved lipase Shine-Dalgarno sequence and lacks a PstI site immediately downstream from the cat gene.
3.2. DNA preparation and transformation DNA-preparation, transformation and cloning procedures have been described elsewhere . 3.3. PCR-mutagenesis Site-directed mutagenesis was performed by using the overlap extension method in combination with the polymerase chain reaction . The SalI-EcoRI fragment and the EcoRI-PstI fragment were amplified for mutagenesis of positions 269 and 369, and positions 559 and 600, respectively. Synthetic oligonucleotides were synthesized by the phosphoamidite method in a DNAsynthesizer (model 380B; Applied Biosystems, Forster City, CA). The DNA sequences of all mutants were analysed by using the dideoxynucleotide termination method of Sanger et al. . 3.4. Agar-plate assay Lipase activity on various substrates was determined by an agar-plate assay with tributyrin-basic agar (Merck, Darmstadt, FRG) containing either 1% Tween 20, 1% Tween 80 or 1% tributyrin as substrate. Phospholipase activity was determined using egg yolk as substrate (Merck, Darmstadt, FRG). 3.5. Spectrophotometrical determination of lipase activity Quantitative lipase activity was determined spectrophotometrically at 405 nm by monitoring hydrolysis of the substrate p-nitrophenylcaprylate (1.3 mg/ml). The buffer consisted of 20 mM Tris" HC1 (pH 9.0), 10 mM CaC12, and 0.1%Triton X-100. 3.6. SDS-PAGE of the supernatant The supernatants of the cultures were lyophilized, resuspended in 1/30 volume double dis-
tilled H20 and dialysed. Proteins were analysed by SDS-PAGE  and stained with Coomassie brillant blue G-250. 3. 7. Western blotting
After SDS-PAGE, proteins were electrophoretically transferred to Hybond-ECL (nitro-cellulose, NC) membrane (Amersham, Braunschweig, FRG) . The lipase and its degradation products were detected with rabbit anti-lipase antibodies, a generous gift from Dr. K.L. Schimz, KFA Jfilich, FRG.