The lipase gene, geh, from Staphylococcus aureus NCTC8530 was cloned in Staphylococcus carnosus. DNA sequencing revealed an open reading frame (ORF) of 2046 nucleotides encoding a 682-amino-acid protein with a molecular mass of 76900 Da. Determination of the transcriptional start site revealed a 203-nucleotide mRNA leader. Expression of geh in the protease-negative S. carnosus (pT181copSA22) resulted in overexpression of a 83-kDa lipase found in the culture supernatant. N-terminal protein sequencing and sequence comparison with three other staphylococcal lipases suggest that this lipase is organised as a pre-pro-enzyme. The substrate specificity of this lipase is different from the Staphylococcus hyicus lipase. The S. hyicus lipase expressed both a high Ca(2+)-dependent phospholipase and lipase activity while the S. aureus lipase lacked this phospholipase activity and its activity with tributyrylglycerol or p-nitrophenyl octanoate is hardly stimulated by Ca2+ ions. A hybrid protein was constructed in which the C-terminal 146 residues of the S. hyicus lipase were substituted by 145 residues of the C-terminal of the S. aureus lipase, which contains the proposed active-site amino acids Asp602 and His641. The hybrid enzyme was still active and revealed an intermediary enzymic activity. The most striking effect was that it had lost the S. hyicus-specific phospholipase activity and that, in contrast to the two parental enzymes, its activity with p-nitrophenyl octanoate became highly sensitive to the presence of Ca2+. These observations suggest that the C-terminal domain of the S. hyicus lipase strongly contributes to the binding pocket of the polar headgroup of phospholipids. The Ca(2+)-binding site seems to be located in the N-terminal fragment of the S. hyicus lipase. The fact that two closely related enzymes differ in the need for Ca2+ underscores the notion that it plays a structural rather than a catalytic role.

The lipase gene, geh, from Staphylococcus aureus NCTC8530 was cloned in Staphylococcus carnosus. DNA sequencing revealed an open reading frame (ORF) of 2046 nucleotides encoding a 682-amino-acid protein with a molecular mass of 76900 Da. Determination of the transcriptional start site revealed a 203-nucleotide mRNA leader. Expression of geh in the protease-negative S. carnosus (pT181copSA22) resulted in overexpression of a 83-kDa lipase found in the culture supernatant. N-terminal protein sequencing and sequence comparison with three other staphylococcal lipases suggest that this lipase is organised as a pre-pro-enzyme. The substrate specificity of this lipase is different from the Staphylococcus hyicus lipase. The S. hyicus lipase expressed both a high Ca(2+)-dependent phospholipase and lipase activity while the S. aureus lipase lacked this phospholipase activity and its activity with tributyrylglycerol or p-nitrophenyl octanoate is hardly stimulated by Ca2+ ions. A hybrid protein was constructed in which the C-terminal 146 residues of the S. hyicus lipase were substituted by 145 residues of the C-terminal of the S. aureus lipase, which contains the proposed active-site amino acids Asp602 and His641. The hybrid enzyme was still active and revealed an intermediary enzymic activity. The most striking effect was that it had lost the S. hyicus-specific phospholipase activity and that, in contrast to the two parental enzymes, its activity with p-nitrophenyl octanoate became highly sensitive to the presence of Ca2+. These observations suggest that the C-terminal domain of the S. hyicus lipase strongly contributes to the binding pocket of the polar headgroup of phospholipids. The Ca(2+)-binding site seems to be located in the N-terminal fragment of the S. hyicus lipase. The fact that two closely related enzymes differ in the need for Ca2+ underscores the notion that it plays a structural rather than a catalytic role.

arsR, the first gene of the Staphylococcus xylosus (pSX267) arsenic/antimonite resistance (ars) operon encodes a negative regulatory protein, ArsR, which mediates inducibility of the resistances by arsenic and antimony compounds. ArsR, which has no obvious DNA-binding motif in its primary structure, was purified from an ArsR-overproducing Escherichia coli strain and identified as a DNA-binding protein by its behaviour in gel mobility shift assays. ArsR had a specific affinity for a 312 bp DNA restriction fragment carrying the ars promoter; the minimum sequence complexed by ArsR was a 75 bp polymerase chain reaction (PCR) fragment, which mainly comprised the -35 and -10 regions of the promoter. The effect of inducers on the DNA-binding activity of ArsR was examined by in vitro induction assays; only arsenite inhibited DNA-binding of the repressor. DNase I footprinting revealed two protected regions within the promoter region, spanning 23 and 9 nucleotides, respectively. Furthermore, a new cleavage site for DNase I between the protected regions was made accessible by binding of the repressor. The footprints cover a region of three inverted repeats located between the -35 and -10 motifs of the ars promoter. By high resolution footprinting with the hydroxy radical, five sites of close contact between the protein and DNA were identified.

An esterase of Streptomyces diastatochromogenes was purified to homogeneity from culture filtrate. The purified enzyme had a molecular mass of 30,862 ؎ 5.8 Da, as determined by electrospray mass spectrometry. The esterase-encoding gene was cloned on a 5.1-kb MboI fragment from S. diastatochromogenes genomic DNA into Streptomyces lividans TK23 by using plasmid vector pIJ702. Nucleotide sequence analysis predicted a 978-bp open reading frame, estA, encoding a protein of 326 amino acids, a potential ribosome binding site, and a putative 35-or 36-residue signal peptide for secretion in S. lividans or S. diastatochromogenes, respectively. The transcriptional initiation site was mapped 29 nucleotides upstream from the predicted translational start codon of estA in S. diastatochromogenes. The protein sequence deduced from the estA gene was similar to that of the esterase from the plant pathogen Streptomyces scabies. Both enzymes lacked the conserved motif GXSXG carrying the active-site serine of hydrolytic enzymes. A serine modified by [1,3-3 H]diisopropyl fluorophosphate was located at position 11 of the mature enzyme in the sequence GDSYT. This finding and results obtained by site-directed mutagenesis studies indicate that serine 11 may be the active-site nucleophile.Streptomycetes are gram-positive, saprophytic soil microorganisms that use a wide variety of extracellular hydrolytic enzymes, including chitinases, cellulases, xylanases, proteases, lipases, and nucleases (50), to degrade organic material in the soil. Polysaccharidases, proteases, and enzymes exhibiting unusual catalytic activities have been studied extensively (35). However, except for phospholipase D, only a few streptomycete lipolytic enzymes and their corresponding genes have been analyzed so far, although Sztajer et al. (46) reported high lipolytic activity in Streptomyces strains. An esterase secreted by the plant-pathogenic strains of Streptomyces scabies has been characterized at the protein (31) and DNA (40) levels. The enzyme is believed to be important in pathogenicity and penetration of S. scabies by hydrolyzing ester bonds in suberin, a waxy polyester covering the external portions of the plants. Recently, genes encoding extracellular lipases from Streptomyces sp. strain M11 (36) and Streptomyces albus G (17) have been cloned and sequenced.Esterases and lipases are carboxylic ester hydrolases (EC 3.1.1). The carboxylesterases (EC 3.1.1.1) hydrolyze water-soluble or emulsified esters with relatively short fatty acid chains, whereas lipases (triacylglycerol acyl hydrolases; EC 3.1.1.3) preferentially act on emulsified substrates with long-chain fatty acids. The hydrolytic mechanism of most of the known esterases and lipases resembles that of serine proteases. These hydrolases all contain a similar catalytic triad, generally consisting of a nucleophilic serine residue that acts in conjunction with a histidine and an aspartic acid residue (8,10,18,51). Microbial lipolytic enzymes have become biotechnologically important enzymes used for hydrol...