engleski

3D-Structure features of novel GDSL-enzyme by bioinformatic methods and mass spectrometry

Lipases (triaclyglycerol acylhydrolases, EC 3.1.1.3) catalyse hydrolysis and synthesis of lipids, depending on the reaction conditions. Their natural substrates are insoluble in water ; therefore hydrolysis takes place on lipid-water interface. This property distinguishes lipases from classical esterases. The ability of these enzymes to stereospecifically catalyse various reactions on a broad range of substrates gives them significant biotechnological potential. Native extracellular lipase from Streptomyces rimosus (SrL) was purified and biochemically characterised. Also, the gene for this enzyme was cloned and primary structure of the protein deduced from nucleotide sequence. Although SrL was shown to be a true lipase by its activity characteristics, sequence homology searches showed no overall amino acid sequence similarity to other lipases in databases, and classified this protein in family II of bacterial lipolytic enzymes (GDSL-hydrolases). Similarity (~25%) was found with the Streptomyces scabies esterase (SsE), which is the only member of family II with solved three-dimensional structure. Secondary structure prediction was performed for SrL by various methods ; consensus prediction was determined and compared to secondary structure of SsE. Other structural features were analysed, such as active site and disulfide bridges. Catalytic triad residues were predicted, as well as forming of disulfide bridges. MALDI mass spectrometry (MS) was used to confirm the identity of catalytically active serine by analysis of lipase-inhibitor covalent complex. The pattern of disulfide bridges was also determined with MALDI-MS. High degree of agreement between analysed 3D-structure elements of SsE and SrL suggests that the latter enzyme might have three-dimensional structure different from other lipases.

GDSL-lipase; Streptomyces rimosus; active site; disulfide bridges; structure prediction; bioinformatics; mass spectrometry

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