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An extracellular lipase from Streptomyces rimosus R6-554W has been recently purified and biochemically characterized. In this report the cloning, sequencing, and high-level expression of its gene is described. The cloned DNA contained an ORF of 804 bp encoding a 268-amino-acid polypeptide with 34 amino acid residues at the amino terminus of the sequence that were not found in the mature protein. The theoretical molecular mass (24.172 kDa) deduced from the amino acid sequence of the mature enzyme was experimentally confirmed. This lipase showed no overall amino acid sequence similarity to other lipases in the databases. However, two hypothetical proteins, i. e. putative hydrolases, derived from the genome sequencing data of Streptomyces coelicolor A3(2), showed 66% and 33% identity. In addition, a significant similarity to esterases from Streptomyces diastatochromogenes and Aspergillus terreus was found. Sequence analysis revealed that our novel S. rimosus lipase containing a GDS(L)-like consensus motif belongs to family II of lipolytic enzymes, previously unrecognized in Streptomyces. When the lipase gene was expressed in a S. rimosus lipase-deficient strain harboring the lipase gene on a high-copy-number vector, lipase activity was 22-fold higher than in the original strain.

In a series of four racemic phenoxyalkyl-alkyl carbinols, 1-phenoxy-2-hydroxybutane (1) is enantioselectively acetylated by Burkholderia cepacia (formerly Pseudomonas cepacia) lipase with an E value $ 200, whereas for the other three racemates E was found to be # 4. To explain the high preference of B. cepacia lipase for (R)-(1)-1, a precursor of its transition state analogue with a tetrahedral P-atom, (R P ,S P )-O-(2R)-(1-phenoxybut-2-yl)-methylphosphonic acid chloride was prepared and crystallized in complex with B. cepacia lipase. The X-ray structure of the complex was determined, allowing to compare the conformation of the inhibitor with results of molecular modelling.Keywords: Burkholderia/Pseudomonas cepacia lipase, racemic sec alcohols, transition state (TS) analogue, crystal structure, molecular modelling.Among more than 30 commercially available lipases [1][2][3][4] that are frequently used in enantioselective acylation of alcohols and amines [5,6] or in esterification of carboxylic acids and hydrolysis of their esters [7,8], Burkholderia cepacia (formerly Pseudomonas cepacia) lipase is one of the most thoroughly studied. The X-ray crystal structures of the native [9,10], and inhibited [11,12] enzyme in open conformations were reported. These structural data have been used in computer simulations of enantioselective ester hydrolysis catalysed by B. cepacia lipase [13][14][15]. Other relevant reports comprise the study of electronic effects of substituents on the enantioselectivity of B. cepacia lipase [16] and flexible docking based on structural information of inhibited B. cepacia lipase [17].Models for predicting enantiopreference in lipasecatalysed acylations of sec alcohols have been proposed based on experimental results. Kazlauskas et al. [18] proposed an empirical rule that predicts which enantiomer reacts faster. This rule relates the relative size of the substituents on the stereogenic centre with enantioselectivity. Other authors reported results related to this rule, in particular the effect of large- [19] and medium-sized substituents [20]. However, these rules do not clarify the detailed mechanism of the enantiorecognition.For some time we have been studying lipase catalysed stereoselective acylations [21-24] of conformationally flexible sec alcohols as substrates. Two important results emerged. First, not all lipases acylate macrocyclic, sterically more constrained sec alcohols with higher stereoselectivity compared to their open-chain counterparts [24]. Second, some lipases acylate with increased enantioselectivity acyclic sec alcohols with perturbing L (large) and M (medium) groups at larger distance from the stereogenic centre [23]. In this latter case, a nonmonotonous correlation between E value and the distance (n) of the perturbing groups in 1-4 was observed (Fig. 1).Continuing this project, we have undertaken biocatalytic, structural, and modelling studies to get more defined information on the mechanism that provides a high degree or bias of enantioselectivity in acetylation of ...

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A chemical modification approach combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to identify the active site serine residue of an extracellular lipase from Streptomyces rimosus R6-554W. The lipase, purified from a high-level overexpressing strain, was covalently modified by incubation with 3,4-dichloroisocoumarin, a general mechanism-based serine protease inhibitor. MALDI time-of-flight (TOF) mass spectrometry was used to probe the nature of the intact inhibitor-modified lipase and to clarify the mechanism of lipase inhibition by 3,4-dichloroisocoumarin. The stoichiometry of the inhibition reaction revealed that specifically one molecule of inhibitor was bound to the lipase. The MALDI matrix 2,6-dihydroxyacetophenone facilitated the formation of highly abundant [M + 2H](2+) ions with good resolution compared to other matrices in a linear TOF instrument. This allowed the detection of two different inhibitor-modified lipase species. Exact localization of the modified amino acid residue was accomplished by tryptic digestion followed by low-energy collision-induced dissociation peptide sequencing of the detected 2-(carboxychloromethyl)benzoylated peptide by means of a MALDI quadrupole ion trap reflectron TOF instrument. The high sequence coverage obtained by this approach allowed the confirmation of the site specificity of the inhibition reaction and the unambiguous identification of the serine at position 10 as the nucleophilic amino acid residue in the active site of the enzyme. This result is in agreement with the previously obtained data from multiple sequence alignment of S. rimosus lipase with different esterases, which indicated that this enzyme exhibits a characteristic Gly-Asp-Ser-(Leu) motif located close to the N-terminus and is harboring the catalytically active serine residue. Therefore, this study experimentally proves the classification of the S. rimosus lipase as GDS(L) lipolytic enzyme.