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Characterization of Streptomyces rimosus lipase complexes with inhibitors by kinetics and mass spectrometry

In the last decades hydrolases have attracted great attention as valuable tools for stereospecific hydrolysis and synthesis of many biotechnologically important compounds. The use of lipolytic enzymes has been extended and improved. However, natural enzymes do not always exhibit desired properties (e.g. enantioselectivity) and further steps to optimize them have to be performed. One of the prerequisites for such an optimization is the detailed characterization of the enzyme active site. We have recently identified an extracellular lipase from bacterium Streptomyces rimosus to be the member of GDSL-family of lipolytic enzymes. Bioinformatic analysis further classified it in the poorly characterized family of SGNH-hydrolases. It is suggested that these enzymes have the three-dimensional structures and active sites significantly different from other lipases, suggesting a particular catalytic mechanism. Here we describe the analysis of the active site of Streptomyces rimosus lipase (SrLip) by inhibition with two reagents, 3, 4-dichloroisocoumarine and tetrahydrolipstatine. Although both substances bind to active-site serine and reduce lipolytic activity of SrLip, their inactivation kinetics are different. DCI (30-fold molar excess) almost completely inhibited SrLip already after 15 min of incubation. However, nearly total inhibition of SrL with THL (5560-fold molar excess) was obtained only after 24 h incubation in the presence of 50% 2-propanol. Samples of SrLip inhibited by both studied substances were analyzed by mass spectrometry (MS). The low energy CID multistage MS of a lipase-DCI complex tryptic digest allowed identification of the enzymatically active serine. The release of DCI from the active site was followed by measurement of lipase reactivation and by re-appearing of native lipase peak in the MALDI-TOF mass spectrum. Analysis of the lipase-THL complex with ESI-MS and MALDI-MS showed that approximately 50% of SrLip contains THL covalently bound exclusively to catalytic serine 10. What accounts for the remaining loss of the lipase activity remains to be determined.

lipase; Streptomyces; inhibitors; kinetics; mass spectrometry

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